Systems and methods for detecting a signal and applying thermal energy to a signal transmission element

ABSTRACT

A signal detection system configured for detecting a signal emitted by the contents of a reaction receptacle is also configured to apply thermal energy to a portion of the reaction receptacle to affect a reaction occurring within the reaction receptacle. More particularly, a system for detecting electromagnetic radiation emitted by the contents of a reaction receptacle includes a transmission element configured for transmitting electromagnetic radiation from the contents of the receptacle, a thermal element associated with the transmission element and configured to apply thermal energy to at least a portion of the receptacle, and a detector configured to receive electromagnetic radiation from the transmission element and to generate a signal corresponding to a characteristic of the electromagnetic radiation received by the detector.

This application claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Application No. 61/148,587, filed Jan. 30, 2009, thedisclosure of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to multi-chambered receptacles andassociated instruments and detection devices for use in performingcomplex processes.

BACKGROUND OF THE INVENTION

All documents referred to herein, or the indicated portions, are herebyincorporated by reference herein. No document, however, is admitted tobe prior art to the claimed subject matter.

Highly sophisticated instruments have been developed for performingcomplex assays requiring multiple process steps to be performedsimultaneously and independent of each other. Such instruments can beused to perform chemical analyses, immunoassays, molecular-based tests,and the like. The most advanced of these instruments are capable ofperforming sample-to-result, nucleic acid-based amplification tests(“NAAT”) that allow for walk-away testing. See Friedenberg et al.,“Developing a Fully Automated Instrument for Molecular DiagnosticAssays,” IVD Technology (2005) 11(6):47-53; Hill, “Automating NucleicAcid Amplification Tests,” IVD Technology (2000) 6(7):36-45. Fullyautomated NAAT testing reduces the chances for contamination or usererror and is increasingly important because of a national shortage ofmedical technologists trained to conduct more complex assays, such asNAAT tests. With full automation, the instrument performs all thenecessary steps of an assay with minimal human intervention. For NAATassays, these steps include processing of raw samples to extract one ormore nucleic acids of interest and to separate the nucleic acids frompotentially interfering materials; performing an amplification reaction,such as polymerase-based extension reaction, to increase the sensitivityof the assay (e.g., TMA, SDA or PCR); and detection of the nucleic acidsof interest. In general, however, instruments used to perform NAATassays are not easily portable and their usefulness is typically limitedto large-scale testing in controlled environments. Therefore, a needcurrently exists for a compact system capable of performingsample-to-result, NAAT assays in point-of-use testing, such as in fieldtesting or bedside medical applications.

SUMMARY OF THE INVENTION

The present invention provides compact instruments, detectors andassociated receptacles and processes for performing complex procedures,such as sample-to-result NAAT assays, that permit point-of-use testingat substantial cost savings to conventional, large-scale instrumentsystems. The receptacles include interconnected chambers that can beprepackaged in unit dose form with all of the reagents needed to performan assay. The receptacles are closed systems that minimize opportunitiesfor contamination.

Aspects of the invention are embodied in a system for detectingelectromagnetic radiation from the contents of a receptacle thatincludes a transmission element adapted to transmit electromagneticradiation from the contents of the receptacle, a thermal element inthermal conductivity with at least a portion of the transmission elementand constructed and arranged to apply thermal energy to at least aportion of the transmission element, and a detection element configuredto receive at least a portion of the electromagnetic radiationtransmitted by the transmission element and further adapted generate asignal corresponding to a characteristic of the electromagneticradiation received by the detection element.

In another aspect, at least a portion of the transmission element isconfigured to be in contact with at least a portion of the receptacle.

In another aspect, the transmission element comprises an optic elementadapted to transmit a light emission from the contents of thereceptacle.

In another aspect, the optic element is a transparent or translucentmaterial.

In another aspect, the material is a plastic.

In another aspect, the optic element is adapted to transmit fluorescentlight through at least a portion of the optic element.

In another aspect, the thermal element comprises anelectrically-resistive film secured to a surface of the transmissionelement or embedded in at least a portion of the transmission elementand having an opening therein through which the electromagneticradiation can be transmitted.

In another aspect, the transmission element comprises a peripheral walldefining a cavity, and the thermal element comprises anelectrically-resistive film secured to a surface of the peripheral wallor embedded in at least a portion of the peripheral wall for applyingthermal energy to the space within the cavity.

In another aspect, the thermal element comprises anelectrically-resistive film secured to a surface of the transmissionelement or embedded in at least a portion of the transmission elementand adapted to transmit the electromagnetic radiation through theresistive film.

In another aspect, the electromagnetic radiation is fluorescence and thedetection element is adapted to generate a signal corresponding to theintensity of the fluorescence.

In another aspect, the system comprises a receptacle holding areaconfigured to hold a receptacle, and the transmission element isdisposed adjacent to the receptacle holding area.

In another aspect, a receptacle is disposed in the receptacle holdingarea, and the transmission element is disposed between the receptacleand the detection element.

In another aspect, the transmission element is in contact with thereceptacle.

In another aspect, the system comprises an actuator mechanismconstructed and arranged to move the transmission element between firstand second positions, and the transmission element is in greater contactwith the receptacle in the second position than in the first position.

In another aspect, the transmission element is not in contact with thereceptacle in the first position.

In another aspect, the receptacle comprises a compressible portion thatis at least partially compressed by the transmission element when theactuator moves the transmission element from the first position to thesecond position.

In another aspect, the system includes a thermal element disposedadjacent to the receptacle holding area in opposed relationship to thetransmission element.

According to another embodiment of the invention, a method for detectingelectromagnetic radiation from the contents of a receptacle comprisestransmitting electromagnetic radiation from the contents of thereceptacle to a detection element with a transmission element disposedadjacent to the receptacle, applying thermal energy to the transmissionelement to cause a temperature of at least a portion of the transmissionelement to be different from ambient temperature, and detecting theelectromagnetic radiation with the detection element.

In another aspect, the step of applying thermal energy comprises heatinga portion of the transmission element to a temperature above ambienttemperature.

Another aspect includes causing the transmission element to be incontact with at least a portion of the receptacle.

Another aspect includes moving the transmission element between firstand second positions, wherein the transmission element is in greatercontact with the receptacle in the second position than in the firstposition.

In another aspect, the transmission element is not in contact with thereceptacle in the first position.

Another aspect includes compressing at least a portion of the receptaclewith the transmission element.

In another aspect, the transmitting step comprises transmitting a lightemission from the contents of the receptacle.

In another aspect, the detecting step comprises detecting a fluorescentemission from the contents of the receptacle.

In another aspect, excitation energy of a prescribed wavelength isdirected at the contents of the receptacle.

In another aspect, the thermal energy is applied by a thermal element inthermal contact with the transmission element.

In another aspect, the thermal element is in thermal contact with aportion of the transmission element through which the electromagneticradiation is transmitted to the detection element.

In another aspect, the thermal element is in thermal contact with aportion of a transmission element surrounding a portion of thetransmission element through which the electromagnetic radiation istransmitted to the detection element.

In another aspect, the transmission element comprises a peripheral walldefining a cavity, and the thermal element is in thermal contact with atleast a portion of the peripheral wall.

In another aspect, the thermal element is secured to a surface of thetransmission element or embedded in at least a portion of thetransmission element.

In another aspect, thermal energy is applied to the receptacle with athermal element disposed adjacent the receptacle and in opposedrelationship to the transmission element.

In another aspect, detecting the electromagnetic radiation comprisesdetecting electromagnetic radiation of a prescribed wavelength.

In another aspect, detecting the electromagnetic radiation comprisesdetecting the intensity of the electromagnetic radiation.

In another aspect, the contents of the receptacle are maintained at anessentially constant temperature during said method.

In another aspect, the contents of the receptacle are maintained at asubstantially uniform temperature.

In another aspect, an amplification reaction is performed in thereceptacle during the method.

According to another aspect of the invention, a device adapted totransmit electromagnetic radiation and to apply thermal energy to a bodydisposed adjacent to the device comprises a transmission element adaptedto transmit electromagnetic radiation through at least a portion of thetransmission element and a thermal element disposed in thermalconductivity with the transmission element and constructed and arrangedto apply thermal energy to at least a portion of the transmissionelement to thereby raise or lower an outer surface temperature of thetransmission element.

In another aspect, the transmission element comprises an optic elementadapted to transmit a light emission from through at least a portion ofthe optic element.

These and other features, aspects, and advantages of the presentinvention will become apparent to those skilled in the art afterconsidering the following detailed description, appended claims andaccompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C are plan views illustrating a multi-chambered receptacleembodying aspects of the current invention.

FIG. 2 is schematic block diagram of the functional architecture of asystem embodying aspects of the present invention.

FIG. 3 is an exploded perspective view of an automated instrumentembodying aspects of the present invention.

FIG. 4 is a schematic view illustrating an arrangement of compressionpads of a pressure mechanism cluster of the instrument.

FIG. 5 is a plan view of a front side of a door assembly of theinstrument.

FIG. 6 is an exploded perspective view of a fluorometer embodyingaspects of the present invention.

FIG. 7 is a perspective view of a rear housing of the fluorometer.

FIG. 8A is an end view of the rear housing of the fluorometer.

FIG. 8B is a cross-section of the rear housing taken along the line8B-8B of FIG. 8A.

FIG. 8C is a cross-section of the rear housing taken along the line8C-8C of FIG. 8B.

FIG. 9A is an end view of the fluorometer.

FIG. 9B is a cross-section of the fluorometer taken along the line 9B-9Bof FIG. 9A.

FIG. 9C is a cross-section of the fluorometer taken along the line 9C-9Cof FIG. 9B.

FIGS. 10A and 10B are a side and top view, respectively, of anembodiment of a compression pad integrated with a signal detector.

FIG. 11 is a perspective view of an alternative embodiment of acompression pad integrated with a signal detector.

FIG. 12A is a plan view illustrating an alternative embodimentmulti-chambered receptacle embodying aspects of the current invention.

FIG. 12B is an exploded perspective view of the receptacle of FIG. 12A.

FIG. 13 is a front perspective view of an alternative embodiment of anautomated instrument embodying aspects of the present invention.

FIG. 14 is a rear perspective view of the instrument of FIG. 13, with atop portion of an exterior housing removed to show the interior of thehousing.

FIG. 15 is a front perspective view of the instrument of FIG. 13, withthe top portion of the exterior housing removed.

FIG. 16 is a front view illustrating an arrangement of compression padsof a pressure mechanism cluster of the instrument of FIG. 13.

FIG. 17A is a rear perspective view of an air manifold and attachedcomponents of the instrument of FIG. 13.

FIG. 17B is a circuit diagram of the pneumatic system of the instrument.

FIG. 18 is a cross-section of an alternative embodiment of a compressionpad integrated with a signal detector

FIG. 19 is a cross-section of a compression pad integrated with a magnetactuator.

FIG. 20 is an exploded perspective view of a temperature control systemof the instrument of FIG. 13.

FIG. 21 is a schematic view of interconnection circuitry and powersupplies for the fluorometer of FIGS. 6-9C.

FIG. 22 is a schematic view of control, processing and communicationcircuitry for the fluorometer.

FIG. 23 is a schematic view of circuitry for voltage measurement and LEDcontrol for the fluorometer.

FIG. 24 is a schematic view of LEDs, RF shielding, and power filteringcircuitry for the fluorometer.

FIG. 25A is a schematic view of a first front-end amplifier circuit forthe fluorometer.

FIG. 25B is a schematic view of a second front-end amplifier circuit forthe fluorometer.

FIG. 26 is a schematic view of a demodulation circuit for thefluorometer.

FIG. 27 is a graph showing relative fluorescent units detected versustime for a set of manually performed real-time amplification reactions.

FIG. 28 is a graph showing relative fluorescent units detected versustime for a set of real-time amplification reactions carried out usingliquid reagents and receptacles and instruments embodying aspects of theinvention.

FIG. 29 is a graph showing relative fluorescent units detected versustime for a set of real-time amplification reactions carried out using aurine sample, liquid reagents and receptacles and instruments embodyingaspects of the invention.

FIG. 30 is a graph showing relative fluorescent units detected versustime for a set of real-time amplification reactions carried out usingdried reagents and receptacles and instruments embodying aspects of theinvention.

FIG. 31 is a graph showing relative fluorescent units detected versustime for a set of real-time amplification reactions carried out with andwithout oil in the amplification and/or enzyme reagents.

FIG. 32 is a transverse cross-section of a signal transmission element,configured to transmit electromagnetic radiation from a sample containedwithin a receptacle to a detector, incorporated with a thermal elementadapted to apply thermal energy to at least a portion of thetransmission element and configured so as not to impede signaltransmission through the transmission element.

FIG. 33 is an end view of the arrangement shown in FIG. 32.

FIG. 34 is a transverse cross-section of a signal transmission elementincorporated with a thermal element disposed on an inner wall of achamber defined within the transmission element and configured so as notto impede signal transmission through the transmission element.

FIG. 35 is a transverse cross-section of a signal transmission elementincorporated with a thermal element disposed on an inner end face of thetransmission element and adapted to transmit electromagnetic radiation.

FIG. 36 is a partial transverse cross-section of a signal transmissionelement incorporated with a thermal element and a reaction receptacledisposed between an end of the transmission element and a thermalconductive element of the temperature control system, wherein the signaltransmission element is configured to function as a compression pad andis shown in a first, non-compressing position.

FIG. 37 is partial transverse cross-section of the signal transmissionelement of FIG. 36, with the signal transmission element shown in asecond position compressing a chamber of the reaction receptacle againstthe thermal conductive element.

FIG. 38 is a schematic view of a control circuit for a thermal elementincorporated within a transmission element.

GENERAL OVERVIEW OF THE INVENTION

The present invention relates to multi-chambered receptacles that can beused to perform one or more manual or automated processes with a sampleof interest, such as determining the chemical composition of asubstance, measuring the activity of a group of metabolic enzymes, ortesting for the presence of one or more analytes in a sample. Some orall of the chambers of the receptacles are interconnected by means ofblocked or sealed passages that can be permanently or temporarily openedto permit the movement of substances between chambers. The arrangementof chambers within the receptacles permits complex processes to beperformed by allowing different steps of a process or multiple processesto be performed non-sequentially and/or simultaneously.

Receptacles of the present invention may be constructed of flexible orrigid materials, as well as combinations thereof, provided thereceptacles permit substances to be forced or drawn between chambers. Atleast some of the chambers can be pre-loaded with process materials(e.g., reagents) and then enclosed or sealed off from the environmentprior to loading sample material. The process and sample materials maybe comprised of liquids, solids, gases, or combinations thereof. Oncesample material is loaded into a chamber or chambers, the receptacle maybe sealed or otherwise closed to maintain all materials within thereceptacle during a procedure. Alternatively, a sample chamber mayremain open after a procedure has been initiated so that some or all ofthe sample material is added to the receptacle after the procedure hasbegun.

The passages interconnecting the chambers are sized and arranged topermit substances to pass between adjacent chambers. The substances arepreferably fluids or fluidized substances and may include, for example,gels, emulsions, suspensions and solids, where the solids may betransported through the passages alone or using, for example, a fluidcarrier, such as an inert oil. Barriers are provided to block themovement of substances through the passages until such movement isdesired. The receptacles, or the receptacles in cooperation with anautomated instrument, can include one or multiple types of barriers.Such barriers may be constructed from the materials of the receptacle(e.g., openable seals), or they may be fixed, movable or alterablecomponents or substances positioned adjacent to or inserted into thepassages (e.g., valves, magnetically-responsive particles, orheat-sensitive wax plugs), or they may be components of the automatedinstrument that apply a reversible, compressive force to the passages(e.g., actuators).

Altering or removing a barrier between adjacent chambers allows asubstance present in one chamber to be forced or drawn into an adjacentchamber. This movement may be achieved by, for example, the action of apressure source, such as an actuator or a group of actuators that areadapted to apply pressure to the exterior of the chamber to therebycollapse or partially collapse the chamber to force all or a portion ofthe material between chambers. The material may be movedunidirectionally or bidirectionally between chambers where, for example,a mixing of combined materials is desired.

The chambers are arranged in the receptacle so that there are at leasttwo non-linear paths that allow for steps of a process to be performedindependent of each other. This provides a tremendous advantage in thatthe substances of two or more sets of chambers can be mixed or combinedbefore the resulting mixtures or combinations, or the underlyingsubstances of the separate sets of chambers, are contacted with eachother. By permitting process steps to be performed independent of eachother, complex procedures having a series of steps that cannot or arepreferably not performed linearly (i.e., steps are performedsequentially) can be performed with the receptacles of the presentinvention.

The compact design of the receptacles and systems of the presentinvention makes them especially suitable for use in point-of-care andfield applications. By sealing off chambers pre-loaded with the processmaterials needed to carry out a process, contamination and user errorissues are substantially minimized. The receptacles of the presentinvention are also ideal for unit dose testing, where chambers of thereceptacles are pre-loaded with the precise amounts of process materialsrequired to conduct a test.

DETAILED DESCRIPTION OF THE INVENTION

While the present invention may be embodied in a variety of forms, thefollowing description and accompanying figures are merely intended todisclose some of these forms as specific examples of the presentinvention. Accordingly, the present invention is not intended to belimited to the forms or embodiments so described and illustrated.

DEFINITIONS

The following terms have the following meanings unless expressly statedto the contrary. It is noted that the term “a” or “an” entity refers toone or more of that entity; for example, “an analyte,” is understood torepresent one or more analytes. As such, the terms “a” or “an,” “one ormore,” and “at least one” can be used interchangeably herein.

Adjacent/Adjacently. With reference to chambers, the term “adjacent” or“adjacently” means that the referred to chambers adjoin each other(i.e., the chambers are positioned directly next to each other in areceptacle). The only structure separating adjacent chambers of areceptacle when a process is initiated is a seal. Thus, adjacentlypositioned chambers are not connected to each other by channels orpassageways.

Ambient Temperature. By “ambient temperature” is meant the temperatureof a surrounding environment, which may include a fluid (e.g., air orliquid) or solid structure.

Amplification/Amplification Reaction. By “amplification” or“amplification reaction” is meant a procedure for increasing the amount,concentration or detectability of a substance that is indicative of thepresence of an analyte in a sample.

Amplification Conditions. By “amplification conditions” is meanttemperature conditions adequate to effect an amplification reaction.

Amplification Oligonucleotide. By “amplification oligonucleotide” ismeant an oligonucleotide that binds to a target nucleic acid, or itscomplement, and participates in a nucleic acid-based amplificationreaction.

Amplification Product. By “amplification product” is meant a nucleicacid generated in a nucleic acid-based amplification reaction thatcontains a target sequence for detection.

Amplification Reagent. By “amplification reagent” is meant a materialcontaining one or more components needed for an amplification reaction.In a nucleic acid-based amplification reaction, such components mayinclude amplification oligonucleotides (e.g., primers and/orpromoter-primers), nucleoside triphosphates, and/or cofactors needed foramplification of a target nucleic acid sequence (e.g., divalent cationssuch as Mg⁺⁺).

Analyte. By “analyte” is meant a sample, or a component of a sample,that is undergoing analysis.

Assay. By “assay” is meant a qualitative or quantitative analysis of oneor more analytes.

Barrier. By “barrier” is meant a structure or material that impedes orprevents the movement of substances between spaces.

Blocked. By “blocked” is meant closed to the movement of a substance.

Binding Agent. By “binding agent” is meant a molecule or molecularcomplex capable of binding to a component of a sample or reactionmixture. The binding agent may be, for example, an antibody, antigen,peptide, protein, nucleic acid or analog thereof, organic molecule, orcomplex of any of the foregoing (e.g., antibody:nucleic acid complex).

Burstable Seal. By “burstable seal” is meant a seal that ruptures orpeels when sufficient pressure is applied to the seal.

Capture Agent. By “capture agent” is meant a binding agent capable ofbinding to an analyte and of being directly or indirectly bound to asolid support.

Capture Probe. By “capture probe” is meant a binding agent capable ofbinding to a nucleic acid analyte.

Chamber. By “chamber” is meant a distinct section or space within areceptacle.

Chamber-Defining Member. By “chamber-defining member” is meant the whole(e.g., bladder) or a part (e.g., wall) of what determines the volume ofa chamber. The chamber-defining member may consist of a single component(e.g., one layer) or multiple components (e.g., a plurality of layersbonded together).

Closed/Closing. With reference to a chamber, “closed” or “closing” meansthat a chamber of a receptacle is not in fluid communication, or thechamber is placed in a condition in which it is not in fluidcommunication, with any other chamber of the receptacle. With referenceto a sample-holding receptacle, “closed” means that all chambers of areceptacle are maintained in a substantially airtight environmentrelative to the ambient environment. With reference to a receptacleprior to sample addition, “closed” means that all chambers of areceptacle except a sample-receiving chamber are maintained in asubstantially airtight environment relative to the ambient environment.

Concentrate. By “concentrate” is meant to limit dispersion of one ormore components within a chamber.

Contiguous Path of Chambers. By “contiguous path of chambers” is meant aseries of adjacently connected chambers.

Directly Connected. By “directly connected” is meant that there are nointervening chambers in the connection between two referred to chambers.

Distinct Connection. By “distinct connection” is meant a connection thatis separate from and non-overlapping with any other connection of areceptacle.

End Chambers. By “end chambers” is meant the outermost chambers of alinear path of chambers.

Enzyme Reagent. The phrase “enzyme reagent” refers to a material thatcontains at least one enzyme that participates in a process. In anucleic acid-based amplification reaction, the enzyme reagent maycontain one or more enzymes that catalyze the synthesis of DNA and/orRNA polynucleotides using an existing strand of DNA or RNA as atemplate. Examples of such enzymes include DNA-dependent DNA polymerases(e.g., DNA polymerase I from E. coli and bacteriophage T7 DNApolymerase), DNA-dependent RNA polymerases or transcriptases (e.g.,DNA-dependent RNA polymerases from E. coli and bacteriophages T7, T3 andSP6), RNA-dependent DNA polymerases or reverse transcriptases, andRNA-dependent RNA polymerases (e.g., an RNA replicase).

Essentially Constant Temperature. By “essentially constant temperature”is meant a temperature that does not vary temporally by more than arelatively small amount (i.e., not more than 0.5° C.).

Flexible. By “flexible” is meant a property of a material that allows ityield to a reasonable force without tearing or breaking.

Fluid. By “fluid” is meant a substance that tends to flow or to conformto the shape of its receptacle (e.g., a liquid or gas). The fluid may bea fluidized substance or mixture of liquids or gases, such as anemulsion. As used herein, the term “fluid” also refers to a substance,such as a paste, that yields to pressure by changing its shape.

Fluidized. By “fluidized” is meant a substance that has been altered sothat it is in a form or medium that has fluid characteristics.

Immiscible Fluid. By “immiscible fluid” is meant a fluid that does notmix with one or more liquids contained in a receptacle.

Immunoassay. By “immunoassay” is meant an assay which involves anantibody-antigen interaction.

Independently Combining. The phrase “independently combining” meansseparately combining two or more sets of substances in distinct chambersof a receptacle, where the separate combinations of substances do notcome into contact with each other.

Indirectly Connected. By “indirectly connected” is meant that there areone or more intervening chambers in the connection between two referredto chambers.

Interconnected. The term “interconnected” refers to chambers that arefluidly connected or connectable, as in the case of an openableconnection.

Intermediate Between. The phrase “intermediate between” means that thereferenced chamber is located between and in the same linear path aseach of two other identified chambers, or that the referenced chamber islocated between and in a different linear path with each of two otheridentified chambers.

Intermediate Chamber. By “intermediate chamber” is meant a chamber thatis connected by openable connections to at least two other chambers.

Isolated. By “isolated” is meant that one or more components of a sampleare sequestered from one or more other components of the sample.

Label. By “label” is meant any substance having a detectable property.

Linear Path. By “linear path” is meant a contiguous path ofconsecutively ordered chambers interconnected by a plurality of openableconnections and defined by a first end chamber, a last end chamber, andone or more intermediate chambers disposed between the first and lastend chambers.

Non-Circular Arrangement. The phrase “non-circular arrangement” refersto an arrangement of interconnected chambers that includes two or morelinear paths, where the end chambers of the linear paths are notcircularly arranged about a central chamber.

Non-linear. By “non-linear” is meant at least two contiguous paths ofconsecutively ordered chambers that share less than all chambers incommon.

Non-sequential. By “non-sequential” is meant that certain steps of aprocess are performed independent of each other rather than in sequence.

Nucleic Acid-Based Amplification. By “nucleic acid-based amplification”is meant an amplification reaction that is dependent upon the presenceof a nucleic acid.

Oligonucleotide. By “oligonucleotide” is meant a polymeric chain of atleast two, generally between about five and about 100, chemicalsubunits, each subunit comprising a nucleotide base moiety, a sugarmoiety, and a linking moiety that joins the subunits in a linear spatialconfiguration. Common nucleotide base moieties are guanine (G), adenine(A), cytosine (C), thymine (T) and uracil (U), although other rare ormodified nucleotide bases able to hydrogen bond are well known to thoseskilled in the art. Oligonucleotides may optionally include analogs ofany of the sugar moieties, the base moieties, and the backboneconstituents. Preferred oligonucleotides of the present invention rangein size from about 10 to about 100 residues. Oligonucleotides may bepurified from naturally occurring sources, but preferably aresynthesized using any of a variety of well-known enzymatic or chemicalmethods.

Openable Connection. By “openable connection” is meant a passage thatcan be temporarily or permanently altered from a “closed” state in whichthe connection is blocked by a barrier to an “open” state in which thebarrier has been modified or moved so that a substance can pass throughan opening in the passage.

Optically Transmissive. The phrase “optically transmissive” is areference to materials permitting the passage of light, so that lightemitted on one side of the materials is detectable by an optical devicepositioned on the opposite side of the materials.

Primer. By “primer” is meant an amplification oligonucleotide capable ofbeing extended at its 3′-end in the presence of a polymerase in atemplate-dependent manner.

Probe. By “probe” is meant a binding agent that binds to an analyte orother substance in a reaction mixture to form a detectable probe:targetcomplex indicative of the presence of the analyte in a sample under theconditions of a process. For nucleic acid-based reactions, the probecomprises an oligonucleotide having a base sequence sufficientlycomplementary to a nucleic acid sequence indicative of the presence of atarget nucleic acid to form a detectable probe:target complex therewith.A probe may also include non-complementary sequences, such as a sequencefor immobilizing the probe on a solid support, a promotor sequence, abinding site for RNA transcription, a restriction endonucleaserecognition site, or sequences which will confer a desired secondary ortertiary structure, such as a catalytic active site or a hairpinstructure, which can be used to facilitate detection and/oramplification. Probes of a defined sequence may be produced bytechniques known to those of ordinary skill in the art, such as bychemical synthesis, and by in vitro or in vivo expression fromrecombinant nucleic acid molecules.

Process. By “process” is meant a series of actions, changes or functionsperformed on or with a substance to bring about a result.

Purified. By “purified” is meant that one or more components of a sampleare removed from one or more other components of the sample.

Reagent. By “reagent” is meant any non-sample substance used in aprocess, including reactants in a chemical, biochemical or biologicalreaction, diluents, solvents, wash materials, rinse materials, buffersand the like.

Real-Time. The phrase “real-time” means that a characteristic of areaction is or is capable of being detected as the reaction isoccurring.

Receptacle. By “receptacle” is meant a device having a plurality ofinterconnected chambers capable of receiving and/or holding substances.

Reconstitution Reagent. By “reconstitution reagent” is meant a reagentused to alter a non-fluid process material to a fluid or fluidizedstate.

Sample. By “sample” is meant a substance capable of being subjected, inwhole or in part, to a process.

Sample Processing Reagent. By “sample processing reagent” is meant areagent that alters or is useful for altering the original state of asample.

Sample Receiving Chamber. By “sample receiving chamber” is meant achamber of a receptacle that is open or openable for receiving a sampleto be processed.

Seal. By “seal” is meant a barrier formed between adjacent chambers. Theseal may be, for example, a heat seal formed between opposedthermoplastic sheets.

Substantially Uniform Temperature. By “substantially uniformtemperature” is meant a temperature that does not vary spatially by morethan a relatively small amount.

Target Nucleic Acid. By “target nucleic acid” is meant a nucleic acidanalyte.

Target Sequence. By “target sequence” is meant a nucleic acid sequencecontained within a target nucleic acid or its complement that isamplified and/or detected in a detection assay.

Water Vapor Transmission Rate. By “water vapor transmission rate” or“WVTR” is meant the steady state rate at which water vapor permeatesthrough a material at specified conditions of temperature and relativehumidity. Water vapor transmission rate values are expressed in g/m²/24hrs. A PERMATRAN-W® water vapor permeation instrument available fromMOCON, Inc. of Minneapolis, Minn. (Model 3/33) can be used to measureWVTR in accordance with ASTM F 1249.

Multi-Chambered Receptacles

Receptacles in accordance with the present invention include a pluralityof interconnected, non-linearly arranged chambers arrayed to perform oneor more processes. The precise dimensions of the receptacles will dependupon the number and arrangement of the chambers, as well as the volumeof substances to be loaded or moved into the chambers. The receptaclespreferably have relatively broad surface dimensions in relation to theirthicknesses, although this is not a requirement. The receptacles arepreferably formed from top and bottom portions, each of which may bemade with rigid and/or flexible materials. In a preferred mode, at leastone of the top and bottom portions of the preferred receptacles is atleast partially flexible.

The non-linear arrangement of chambers allows for non-sequentialprocessing of samples in the receptacles. The exact number,configuration, sizes and arrangement of chambers will depend on theparticular process or processes to be performed. Distinct from thesurrounding receptacle materials, the chambers may be constructed ofrigid or flexible materials or combinations thereof. Material selectionwill depend in part on whether the chambers must yield to an externalpressure in order to move substances between chambers. The chambers maybe of any shape that does not interfere with the movement of substancesbetween chambers, which includes generally planar or bubble-like shapes,including hemispherical and spherical shapes. In a preferred embodiment,the chambers are generally flat and have a tear-drop shape that funnelssubstances through an open connecting passage and into an adjacentchamber. Tear-drop shaped chambers also advantageously focus pressure onconnecting passages, thereby more readily opening barriers such as sealsand valves used to temporarily block access to adjacent chambers. Thechambers may have the same or different shapes and/or sizes.

Passages used to connect the chambers of a receptacle may be, forexample, portals or passageways that are dimensioned to permitsubstances used in a process to move between chambers. In the case of aportal, a seal or other barrier may be substantially all that separatesadjacent chambers. A passageway on the other hand comprises a conduitextending between chambers. Portals are preferred because they permit amore compact receptacle design and require materials to travel shorterdistances between the various chambers. Some of the passages may remainopen throughout a process, such as a passage leading to a waste chamber,while others are blocked by barriers until it is desired to move asubstance between chambers. The barriers can be selectively altered from“closed” to “open” states to create substance-transferring connections,which are preferably fluid-transferring connections for moving fluidsand substances in a fluidized form. A barrier may be an external force,such as a compressive force provided by, for example, a clamping device(e.g. pneumatically driven actuator having a clamping pad), or it may bea seal or valve that yields to pressure, is mechanically operated, or isaltered by, for example, heat, laser ablation, or a chemical orbiochemical reaction to provide an opening between chambers, or it maybe an external force/seal combination. In a preferred embodiment, thepassages are blocked with a heat seal, such as a V-shaped or chevronseal, that is reinforced with a compressive force during use. While theblocking properties of some barriers are designed to be affected byconditions such as heat (e.g., wax plugs), or may be affected by achemical agent moved into or formed in a chamber, barriers formed orpositioned in a receptacle should not otherwise be influenced byenvironmental conditions or substances that are contained within thechambers.

Once a barrier separating adjacent chambers has been removed or alteredto create an opening, a substance may be pushed, pressed, drawn orotherwise moved, such as by gravity, into a neighboring chamber. If thechambers are to be acted upon by a pressing force, such as a roller oran actuator-driven compression pad, to move substances between chambers,then the chambers are formed to have at least one flexible surface thatyields to the pressure of the pressing force. Otherwise, the chambersmay be constructed of a rigid material, as in the case of pads orvacuums used to push or draw substances into adjacent chambers. This isalso true where gravity draws a substance from a chamber that ispositioned above a receiving chamber, although in some applications itis desirable for both chambers to have at least one flexible surface sothat substances can be moved back-and-forth between chambers in order tomix two substances.

The chambers are arranged in the receptacles so that there are at leasttwo distinct linear paths. Each of the paths includes at least threecontiguously connected chambers, with at least one of the pathspreferably including five or more contiguously connected chambers. Asused herein, the phrase “contiguously connected chambers” refers to aseries of directly connected chambers in which the chambers aresuccessively arranged, with one chamber coming after another. Each ofthe paths may share at least one but less than all chambers in commonwith any other path in the receptacle. This arrangement of chambersallows certain steps of a process to be performed independently and/orsimultaneously. In this way, substances used in a complex process can beprepared and kept segregated until it desirable to combine them. Suchindependent activities may include, for example, dissolving, diluting,mixing, combining or reacting substances. By way of example only, oneset of adjacent chambers may contain a dried, primer-containingamplification reagent for use in amplifying a target nucleic acidsequence present in a sample and a reagent for reconstituting theamplification reagent, while another set of adjacent chambers maycontain a dried enzyme/probe reagent for use in amplifying and detectingthe target nucleic acid sequence and a reagent for reconstituting theenzyme reagent. If, in this particular example, the amplification andenzyme/probe reagents are prematurely combined in their reconstitutedforms, there is some risk of target-independent amplification that couldinterfere with the amplification of the target or consume scarcereagents for amplification. See, e.g., Adams et al., “Decoy Probes,”U.S. Pat. No. 6,297,365. Therefore, it is desirable to separatelyreconstitute these reagents and then to combine them in the presence ofthe target nucleic acid.

For illustration purposes only, FIG. 1C shows a receptacle 10 having anon-linear arrangement of chambers that defines a number of distinctlinear paths. Some of the possible linear paths of this receptacle areidentified with letter designations, each chamber of a linear pathhaving the same letter designation (i.e., A, B, C or D), and eachchamber of a linear path being assigned a distinct number. Thearrangement of the paths is such that the steps of a process can beperformed non-sequentially. For example, a sample material can be addedto chamber A2 and mixed with a binding agent provided from chamber A1before isolating and purifying an analyte in chamber A3. At the sametime or different times, a first dried or solid process material inchamber B2 can be reconstituted with a first reconstitution reagent(e.g., a first solvent) provided from chamber B1. Also, at the same ordifferent times, a second dried or solid process material in chamber B4can be reconstituted with a second reconstitution reagent (e.g., asecond solvent) provided from chamber B5. Finally, the purified analyteand the reconstituted first and second process materials can be combinedin chamber B3 or B4 for detection of the analyte. While there are onlyfour distinct linear paths identified in FIG. 1C, it is readily apparentthat other possible linear paths and combinations of linear paths couldbe utilized.

The non-linear arrangement of chambers in the receptacles can facilitatetheir use in performing multiple processes of the same or differentkind. This is because the chambers can be arranged so that substancesand/or chambers that are not shared between processes remain isolatedduring a process. Thus, the present invention also relates to“universal” receptacles, where a single receptacle can be designed andmanufactured, including pre-loading of process materials, for multipleapplications. In this way, the end-user does not need to have adifferent receptacle for each process to be performed or to predict thevolume requirements for any particular process in advance. Materialsused to form the chambers and barriers of the present invention shouldbe selected to maintain acceptable stability and reactivity levels ofthe various substances used to perform a process. Such materials mayprovide, for example, a moisture barrier for substances that arealtered, degraded or otherwise affected by moisture. In an alternativeor complementary approach, a desiccant, such as calcium oxide, may beloaded with a dried process material to minimize the affects of moisturein a chamber. When a process is being performed in a receptacle, it maybe desirable to sequester the desiccant to prevent it from interferingwith or altering a reaction involving the dried process material. Thedesiccant can be sequestered by, for example, locating it in a sectionof a chamber containing the dried process material or by placing thedesiccant in an adjacent chamber having an open connection with thechamber holding the dried process material. It may be desirable to blockthis open connection after reconstituting the dried process material toprevent unwanted interactions with the reconstituted process material.Yet another approach would be to store the receptacle in a vessel formedfrom a material or materials that provide a moisture barrier and/orwhich includes a desiccant. Common desiccants include clay, silica gels,calcium oxide and synthetic molecular sieves. An example of a molecularsieve is a Type 4A Molecular Sieve Multiform™ Tablet having a 0.45″diameter and a 0.125″ height, where “Type 4A” indicates a pore size of 4angstroms (Multisorb Technologies, Inc., Buffalo, N.Y.; Product No.02-00674AH01).

Similarly, receptacle materials may be selected to protect substancesfrom the environmental affects of exposure to oxygen or electromagneticradiation. Alternatively, the materials used to form the receptacles maybe selected to prevent stored substances from adversely interacting witheach other by selecting materials that provide a barrier againsttransmission of any liquid, solid or gas intended for use in thereceptacle. It may also be desirable to use materials in constructingthe receptacles that protect against evaporation of substances toprevent the activity of those substances from being altered. Further,the materials selected for use should not significantly alter theintended functions of the stored substances, nor should they adhere toor otherwise bind reactants in a manner that significantly affects theirability to participate in a process or processes.

Where a procedure involves sample manipulations that requireconcentrating or moving magnetic particles within or between chambers,at least a portion of the receptacle will need to be constructed ofmaterials that do not substantially interfere with the influence ofmagnetic fields generated by adjacently positioned magnets. Forprocesses requiring heating and/or cooling of all or some of thechambers, either continuously or for precise periods of time, thereceptacles must be capable of an energy transfer on at least one sideof the receptacles that is capable of affecting the thermal conditionsof the contents of chambers requiring heating and/or cooling.Additionally, the receptacles may include optically transparent portionscapable of transmitting light of the visible, infrared and/orultraviolet spectrum to detect changes in the physical characteristicsof a sample, such as color or turbidity, or to enable the detection oflabels that are indicative of the presence of analytes of interest.

Receptacles of the present invention may be constructed from suchmaterials as polymers, glass, silicon, metals, ceramics and combinationsthereof. The materials used will depend, in part, on the means selectedfor moving substances between chambers, whether a physical change in thesample must be visualized or a light signal detected, and the manner inwhich substances present in the receptacles, such as magnetic particles,are to be manipulated. The materials used to construct receptaclesshould be stable under the expected transportation, storage and useconditions. Such conditions include temperature, pressure, humidity andduration of storage. Also, materials used to form flexible chambers ofthe receptacles should yield to the selected pressure forces for movingsubstances between chambers without being torn, punctured or ruptured.

In a preferred embodiment, the receptacles are formed from flexible topand bottom sheets that are of the same or different materials and may bemultilaminates. Each sheet preferably has at least one liquid-imperviouslayer, and the sheets preferably have a relatively uniform thickness,which may be between about 0.05 mm and 2.0 mm. Also, select regions ofone of the multilaminates are preferably optically transparent ortranslucent. The sheets may be formed from, for example, foils—with oneor more holes cut in the foil to provide detection windows asnecessary—and/or thermoplastic materials such as polypropylene (e.g.,Reflex® polyolefins available from the Rexene Corporation, Dallas,Tex.), polyester, polyethylene (e.g., polyethylene teraphthalate (“PET”)and polyethylene naphthalate (“PAN”)), polyvinyl chloride,polyvinylidene chloride, polycarbonate resins (e.g., polyvinyl fluoridefilms) and polyurethane. In a particularly preferred embodiment, the topand bottom sheets are each multilaminates, an example of which is aScotchpak® film layer (3M Corporation, St. Paul, Minn.; Cat. No. ES-48)bonded to a Perflex® foil layer (Perfecseal, Oshkosh, Wis.; Product No.35786). Other suitable materials for forming the flexible sheets of thisembodiment will be appreciated by those skilled in the art. See, e.g.,Burke (1992) WAAC Newsletter 14(2):13-17.

Exemplary laminates include: foil coated PET with Surlyn® blend peellayer (4.5 mils), clear double AlOx coated PET on low-densitypolyethylene (“LPDE”) with coextruded peel layer (4.5 mils), foil coatedLDPE with coextruded peel layer (4.5 mils), foil coated LDPE seal layer(3.5 mils), clear single AlOx coated PET on Biaxial Oriented Polyamidewith peel layer (4 mils), clear AlOx coated PET on LDPE seal layer withzone coat defined frangible seal (2.5 mil), foil barrier with peelsealant. (3.5 mil), clear AlOx coated PET on LDPE seal layer (2.5 mil),clear AlOx coated PET on LDPE seal layer, (4 mil), PET coated Foil withHDPE seal layer, clear AlOx coated PET with EVA based peel layer, (3mil), and foil coated PET with Surlyn® blend peel layer (4.5 mils), aswell as laminates of opaque polyethylene terephthalate (“OPET”), ink,white LDPE, aluminum foil, polyethylene (“PE”), linear low-densitypolyethylene (“LLDPE”), and nylon and OPET, white PE, foil, adhesive,and EZ Peel® Sealant.

Opposed inner heat sealing layers of the top and bottom sheets in thepreferred receptacles are bonded to form the walls of the chambers andopenable seals (e.g., chevron seals) that separate adjoining chambersusing heat sealing techniques well known in the art. The bonds definingthe walls of the chambers are stronger than the openable sealsseparating chambers so that when pressing forces are applied to thechambers, materials are forced between chambers rather than peelingapart the walls of the chambers. Target seal strengths for chamber sealsmay be on the order of about 9-10 lb/inch, and target seal strengths forpeelable seals may be on the order of 2.2-2.3 lb/inch.

More specifically, flexible or semi-rigid receptacles, orpouches—including features of the receptacles, such as chambers,passages, permanent and semi-permanent (e.g., ruptureable, burstable,peelable, frangible, etc.) seals—can be formed by welding two filmstogether using heated filaments, a heat sealing die, impulse welder, orultrasonic welder or other known techniques. Alternatively, adhesives,or other bonding techniques, capable of forming bonds of differentialseal strengths, can be used.

A receptacle constructed for implementation within the present inventionpreferably includes chambers defined by permanent inter-film bondsformed around the peripheries of the chambers to avoid peeling or creepof the bonds. Semi-permanent seals which are used to initially blockpassages or portals interconnecting adjacent chambers are constructedand arranged to rupture, or burst, when subject to a predetermined,preferably consistent force to provide fluid communication between theadjacent chambers. Application of a compression force to the chambercauses lateral expansion of the fluid or other substance containedwithin the chamber in a direction that is transverse to the direction ofthe force. The permanent and semi-permanent inter-film bonds definingthe chamber preferably have bursting pressures, or seal strengths, suchthat the expanding fluid will generate a sufficient force tohydraulically peel, or rupture, the semi-permanent seal, but will notgenerate force sufficient to peel the permanent bond defining theremainder of the periphery of the chamber. As well known in the art, theburst pressure, or seal strength, of a seal or bond formed by knowntechniques is a function of a number of factors, including, the natureof the materials being bonded together, the temperature at the interfaceof the materials, the pressure applied to the materials, and the dwelltime or period of time during which the assembled films are exposed toelevated temperatures and pressures.

Methods for forming such receptacles having bonds of differential sealstrengths are well-known in the art. Exemplary disclosures can be foundin Johnson et al., “Analytical Test Pack and Process for Analysis,” U.S.Pat. No. 3,476,515 at col. 3, lines 36-56; Freshour et al., “FlexiblePackages Containing Nonfusible High Peel Strength Heat Seals,” U.S. Pat.No. 3,496,061 at col. 2, lines 6-52, and col. 7, lines 50-59; Farmer,“Packaging Device,” U.S. Pat. No. 5,131,760 at col. 4, lines 25-34;Robinson et al., “Diagnostics Instrument,” U.S. Pat. No. 5,374,395 atcol. 31, lines 27-58; and Rees et al., “Method and Apparatus for FormingHeat Seals with Films,” U.S. Pat. No. 6,342,123 (disclosure is directedto the formation of differential seals with a single die).

Surprisingly, it was discovered that there are advantages toconstructing chambers designated for holding moisture-sensitivematerials, such as dried process materials, to include regions thatpermit a greater degree of moisture transmission than surroundingportions of the receptacles. For example, the sheets of the flexiblereceptacle described above may include thermoplastic and foil layers,where at least one of the sheets includes cut-outs in the foil layeraround the chambers containing moisture-sensitive materials. (Cut-outsmay also be needed for chambers requiring light transmission fordetection or other purposes.) To keep the moisture-sensitive materialsdry, the receptacles are placed in sealed, desiccant-containing vessels,where moisture is drawn from chambers holding moisture-sensitivematerials and into the vessels where it is absorbed by the desiccants.The desiccant-containing vessels should be constructed of materialshaving low moisture vapor transmission rates, such as a Mylar® OB12polyester packaging film available from Dupont Packaging and IndustrialPolymers of Wilmington, Del.

To register the receptacles in an instrument, the receptacles may beprovided with attachment holes that are aligned with correspondingmounting posts in the instrument. Alternatively, the receptacles may beprecisely positioned in an instrument using hooks, loops, adhesives andother like attachment materials. Where an instrument includes a slot forreceiving and registering receptacles, those receptacles constructed offlexible materials are preferably supported by a rigid frame about theirperipheries for precisely positioning the receptacles within theinstrument. It is also contemplated that in some embodiments nopositioning structures will be required.

One or more labels or devices providing information that is human and/ormachine readable or recognizable may be affixed to or otherwiseassociated with the receptacles in regions that do not interfere withthe processing of samples. The labels and/or devices may provideinformation relating to the sample type or source and/or the testingprotocol or other process to be run. Markings on labels preferablyinclude scannable barcodes. Such labels may be, for example, peel-offlabels that can be transferred to an associated chart or file.Alternatively, the information may be printed on or formed in a materialused to construct the receptacles.

Substances Used in the Receptacles

The chambers can be loaded with reagents, compounds, compositions orother substances for use in a single process, multiple applications ofthe same process, or multiple processes of the same or different kind(e.g., nucleic acid-based tests and/or immunoassays). The types ofsubstances that can be loaded into the chambers include liquids, solids,gases, and various combinations thereof. For some processes, it may alsobe desirable to leave one or more chambers initially empty so that theymay serve as, for example, sample, waste, venting, mixing or detectionchambers within a receptacle. Receptacles having arrangements ofchambers that can be used to perform any of multiple differentprocedures may have additional empty chambers depending on the number ofprocess materials loaded to perform any particular procedure. Liquidsthat may be loaded and moved between chambers include aqueous andnon-aqueous substances, combinations of liquid substances, such asmixtures of liquid substances and emulsions (with and without anemulsifier or emulsifiers), and liquefied substances, such as solidsmelted by heating or gases condensed by cooling. Solids that may beloaded and moved between chambers include waxes, mixtures of solids, andsolids in liquids, such as suspensions (e.g., colloids, including gels)and slurries. Solids can be in a wide variety of forms, including theirnatural elemental or molecular forms.

Liquid, partially liquid and/or solid substances can be prepared so thatthey are in a dried or altered solid form when loaded into a chamber.Such substances may be the product of, for example, encapsulation,lyophilization, pelletizing, powderizing, tabletization, drying,spotting, including spotting of the same or different substances withina chamber (including multiple spots of the same and/or differentsubstances in array patterns), the formation of particles, fibers,networks or meshes, and absorption and/or drying onto a carrier,including an inner surface of a chamber. These substances may provideadvantages, such as improved stability or durability, enhancedeffectiveness, convenience of manufacturing and handling, preciseamounts of substances, protection against environmental and otherstresses, including temperature, moisture, oxygen and electromagneticradiation, loading into spatially separated sections of a chamber, andprotection against premature and adverse interactions of differentsubstances within a receptacle, including unintended interactionsbetween a sample and process materials.

Solids loaded in the chambers could be useful for such functions asfiltration, immobilization, collection, drying, detection (e.g., probereagents, chromatography, electrophoresis, etc.), and amplification(e.g., amplification oligonucleotides and enzyme reagents). A solid mayremain unchanged during a process or it may be altered prior to or afterinitiating a process. Types of alterations may include dissolution, theformation of a suspension, slurry or gel, melting, or a chemical,biochemical or biological reaction. Such alterations may be caused by,for example, an interaction with a fluid loaded or formed in an adjacentchamber, heating, cooling, irradiating, sonicating and/or subjecting thesolid or solids of a chamber to an electrical current or magnetic field.

Substances may be loaded into the receptacles using manual,semi-automated or automated methods. Particular process materials thatmay be loaded into the receptacles include, for example, dried and/orliquid reagents, including binding reagents (e.g., nucleic acids,antibodies, antigens, receptors and/or ligands) and signal generators,solvents, diluents, suspensions, solutions, including wash and rinsereagents, and solid supports, including particles, beads and filters.Loading may be accomplished by such means as pouring, pipetting,injecting, spotting, drawing (e.g., applied vacuum or syringe),evacuating, exchanging atmospheres, and the like. Keeping the amount ofair present in a chamber to a minimum is generally preferred and, forreproducibility, the air/material ratios in like chambers should be keptsubstantially uniform across receptacles prepared for identical uses.When loading substances into a flexible receptacle, the substances arepreferably loaded from access openings extending from an edge or edgesof the receptacle into the chambers to be loaded. FIG. 1B provides anillustration of access openings 19, 21, 23, 25, 31, 33, 35 in anexemplary receptacle 10 described more fully below. The opposed sides ofthe receptacle are preferably drawn apart by suctioning to facilitateloading of substances and to control wicking of liquid substances up thesides of partially sealed chambers. Dried or solid substances arepreferably loaded first, and the chambers so loaded are temporarilysealed with a tack seal, which provides a substantially fluid-tight sealto protect substances that are sensitive to or altered by the presenceof moisture. Liquid substances are then loaded, and all openings leadingto chambers with loaded substances are sealed with a heat seal.

Some process materials exhibit a strong tendency to wick up the sides ofthe chambers during loading and, in some instances, migrate intoadjacent chambers where they can alter the nature, concentration and/orperformance of other process materials. By way of example, ifamplification and enzyme reagents co-mingle prematurely, unintendedinteractions could occur which consume a portion of these reagents priorto contacting them with a target nucleic acid. To address this problem,it was discovered that providing oil (e.g., silicone oil) to thechambers prior to loading process materials significantly reduces thewicking effect and improves the performance of processes. Also, when asilicone oil is included in chambers filled near their capacities, anyloss of material during the sealing or closing process is typicallylimited to the inert, inactive oil rather than active process materials.Further, an oil layer situated above a heat-labile process material(e.g., enzyme reagents) will insulate the process material from the hightemperatures used in sealing closed the receptacle.

The use of oil was found to have other benefits as well. For example, ifa process material contains particles or beads (e.g.,magnetically-responsive particles) that tend to settle along the sidesof a chamber, or adjacent passages between chambers, the use of oilhelps to concentrate the particles or beads toward the center of thechamber. Otherwise, it might be difficult to fully re-suspend theparticles or beads or they could clog a passage, thereby preventing orinterfering with the movement of substances between chambers.Additionally, oil can be used to increase the fluid volume of a chamberthat otherwise has an insufficient amount of a process material to fullyor adequately open a barrier in an adjoining passage when pressure isapplied to the chamber. And, because oil is inert, it will not affectthe relative concentrations of combined process materials. Anotherbenefit of oil is that it interferes with the evaporation of liquidsubstances from the chambers. For receptacles including rigid portions,substances may be provided to cavities formed in the rigid portions bysuch means as spraying, spotting or otherwise bonding or adheringsubstances to surfaces of the cavities, or by pouring or pipetting.Alternatively, process materials are provided to the receptacles, inwhole or in part, through resealable openings, such as Luer connections,septums or valves. In this latter embodiment, substances may be added toreceptacles while procedures are in progress.

All substances, except sample material, are preferably provided to thereceptacle and sealed to the environment prior to shipping for use. Bydoing so, processes carried out with the receptacles are easier toperform, opportunities for operator error are minimized, and there isless risk that a receptacle or associated process materials will becomecontaminated. Substances loaded in advance must be provided in a formand kept under conditions such that the substances remain stable priorto use. Among other things, this means that the materials used toconstruct a chamber cannot adversely affect a loaded substance, therebyaltering its intended function or performance. Likewise, a loadedsubstance should not substantially affect the function or performance ofthe chamber it is stored in.

Types of sample materials that can be tested with the receptacles of thepresent invention include both fluid and solid samples. Fluid samplesthat may be tested with the receptacles include, for example, urine,blood, saliva, mucus, seminal fluid, amniotic fluid, cerebrospinalfluid, synovial fluid, cultures, liquid chemicals, condensed gases andwater. Solid samples that can be tested with the receptacles include,for example, tissues, stool, soil, plants, powders, crystals, food andfilters. Sample materials may be provided to the receptacles in a raw orprocessed form. A processed sample is one that has been modified in anymanner, such as by removing components of a raw sample or by otherwisealtering the material from its original state. For example, with a solidsample, it may be necessary to alter the sample either prior to or afteradding the solid material to a sample chamber so that an analyte ofinterest is free to move between chambers of the receptacle. In analtered state, the solid sample may form part of, for example, asuspension, slurry or homogenate or it may be a liquefied or dissolvedform of the solid sample.

Sample materials are preferably introduced into one or more samplechambers of a receptacle through an inlet port immediately prior toinitiating a process, although some of the steps of a process may beinitiated or completed prior to adding sample material to thereceptacle. The inlet port may be an access opening or it may include,for example, the female portion of a Luer connection for insertion of asyringe or other having a male Luer connection. If the sample materialis stable in the receptacle, then the sample material may not need to beadded to the receptacle immediately prior to use. For automated uses ofthe receptacles, it is generally preferable to load sample material intothe sample chamber or chambers of a receptacle manually or in a separateloading device. If sample material is directly loaded into receptaclesbeing held by an associated instrument, there is an increased chance ofcarry-over contamination between receptacles that could lead to a falsepositive or altered result. One such loading device that can be used oradapted for use with flexible receptacles of the present invention isthe FastPack® Sample Dispenser (Qualigen, Inc., Carlsbad, Calif.). Forsome applications, a relatively large volume of sample material may berequired to ensure that there is a detectable amount of an analyte, ifpresent in the sample. Receptacles of the present invention can bedesigned to include sample chambers that are larger than subsequentchambers that are employed to process a sample. Using manually orautomatically activated pressure means, aliquots of a sample can besequentially treated in a neighboring chamber or chambers to removeunwanted components of the sample and to reduce the volume or size ofthe sample being moved between chambers. The unwanted components can betransferred to a designated waste chamber in the receptacle. Separatingan analyte from other components of a sample will generally involveimmobilizing the analyte within a chamber, removing unbound material,and further purifying the immobilized analyte by washing it one or moretimes with a wash reagent.

Instruments for Manipulating the Contents of the Receptacles

Receptacles of the present invention are preferably adapted for use withan automated instrument capable of acting on all or a portion of thechambers of a receptacle to affect the location or state of substancescontained therein. Such actions may include moving substances between orwithin chambers, opening and closing interconnections between chambers,reinforcing barriers between chambers, localized or generalized heatingor cooling, and screening for one or multiple signals or other physical,chemical, biochemical or biological events that may be indicative of,for example, the presence, amount or state of one or more analytes ofinterest. Other effects of such actions may be to mix, combine,dissolve, reconstitute, suspend, isolate, wash or rinse substances of aprocess, to manipulate dried or solid substances, to remove wastesubstances, and/or to reduce the volume of a substance, such as a samplesubstance, to facilitate processing in a receptacle. The instrument maybe used to process substances in a single receptacle or it may beadapted to process substances in multiple receptacles independently andin any desired order, including simultaneously (i.e., parallelprocessing). The instrument, alone or as part of an overall system,preferably has the capability of collecting, analyzing, and/orpresenting data during and/or after a process has been performed. All ora portion of the actions of the instrument are governed by a controller.

The instrument is designed to cooperate with a receptacle to movesubstances between and/or within chambers. Substances may be moved in areceptacle by applying an external pressing force or forces to aflexible surface or surfaces of a chamber, such as by the use of linearactuators or rollers, or by applying an internal pressing force, such asby the use of pistons contained in piston chambers that are in airand/or fluid communication with the contents of selected chambers.Alternatively, a vacuum may be created to draw substances betweenchambers or to different regions of a chamber. Magnetic fields may beused to direct the movement of magnetized substances within and betweenchambers, as well as substances associated therewith. Other means formoving substances between or within chambers may include centrifugalforces, gravitational forces, electrical forces, capillarity,convection, sonication, irradiation and the like. In a particularlypreferred embodiment, one or a combination of actuator pads are used topress substances into adjacent chambers or to move substances withinchambers. The use of a combination of actuators can facilitate serialmovement of substances into adjacent chambers or the mixing ofsubstances within a chamber.

In a preferred mode, burstable heat seals interrupt connecting passagesbetween at least a portion of the adjacent chambers of a receptacle andprovide a barrier against the movement of substances between chambers.Actuator-driven compression pads of a cooperating instrument may be usedto apply pressure to the chambers, thereby peeling the seals apart(e.g., bursting) and allowing some or all of the substances of thechambers to pass into adjacent chambers. Where an opened seal allows fora bidirectional flow of substances, it may be desirable to use at leastone actuator as a clamping device to prevent a backflow of substances.The actuators can also be used as clamps to prevent seals fromprematurely opening. As an example, a chamber may be directly connectedto multiple other chambers. To focus the flow of a substance into adesired chamber, those chambers that are not being utilized aresealed-off by using the actuators as clamps to reinforce sealedinterconnections with the undesired chambers. Actuators may also be usedto control the flow of substances when no seals are provided.

Substances in a chamber, or in multiple chambers, can be mixed in aninstrument by various active and passive means. “Active” methods ofmixing substances involve the application of a mechanical force, such asa pressing force, whereas “passive” methods of mixing substances do notinvolve the application of a mechanical force, such as by gravitation.In one method, substances are mixed by force of flow as a substance ofone chamber is moved into and contacts a substance of a second chamber.By another method, substances are mixed by turbulence when one of thesubstances is forced through the restricted space of a passage joiningtwo chambers. Alternatively, the substances of two chambers can be mixedby forcing the combined substances back-and-forth between the twochambers. Yet another method of mixing involves the use of multipleactuators adjacent a chamber to force combined substances betweendifferent regions of a chamber. In one application of this embodiment,the actuator is a movable optical element of a light detecting device(e.g., fluorometer) that is in sliding engagement with a correspondingring member, where the optical element and the ring member generallyconform to the shape of an associated chamber and move into engagementwith the chamber in an alternating fashion to achieve mixing. In anotherapproach, mixing is carried out by forcing a first substance upward froma first chamber into a second chamber, where it is combined with asecond substance, and then allowing for gravity assisted movement of thecombined substances back into the first chamber. This procedure can berepeated until the desired degree of mixing is achieved. Otherprocedures for mixing may involve, for example, heating and/or coolingto produce convection mixing or sonication, with or without solidparticles.

The instrument and receptacle can also cooperate to manipulatecomponents of a substance. For example, one or more chambers of thereceptacle may include a filter, or series of filters, for removingconstituents of a substance as the instrument actively or passivelycauses the substance to pass through the filter. The constituentsremoved from a substance may include components of a sample materialthat can interfere with a process or solid supports that are used forprocessing a sample material, such as beads, particles, rods, fibers andthe like. These solid supports may be used, for example, to bind ananalyte, either directly or indirectly, or components of a sample. Inanother approach, the instrument may be adapted to cause solid supportsor solid substances in a material to be concentrated in a chamber byimposing a centrifugal force on the receptacle. In an alternativeapproach, magnetically responsive particles present in a chamber can bemanipulated by magnetic forces exerted by a component of the instrument.By isolating the magnetic particles in a specific chamber, substancesbound to the particles remain in the chamber while unbound substancescan be removed from the chamber. In addition to separating wanted fromunwanted materials, solid supports, such as beads and particles, canalso be used in the receptacles of the present invention to facilitate areduction in the volume of a sample material. The initial volume of thesample material may be relatively large to ensure that there is anadequate quantity of the analyte of interest for detection and/orquantification. However, in some cases this initial volume is too largefor practical processing of the sample material in the receptacle.Instruments and receptacles in accordance with the present invention canaddress this problem by immobilizing the analyte on a solid support(e.g., magnetically-responsive particle) in, for example, a samplereceiving chamber, isolating the solid support within the chamber (e.g.,exposing the particles to a magnetic field), and then generating forcesthat remove the remainder of the sample material from the samplereceiving chamber and dispose of it in a designated waste chamber.Alternatively, this same procedure can be performed in an adjoiningchamber of a receptacle having a smaller volume capacity than the samplereceiving chamber by incrementally moving, isolating and separatelyprocessing aliquots of a sample material. By having or moving the solidsupport into a smaller chamber, processing of the sample material may bemore efficient and the consumption of process materials lower.

To purify an analyte, the solid support can be washed one or more timeswith a wash reagent in a designated sample processing chamber. Whenperforming a wash procedure, a force or forces may be imposed by theinstrument which cause the solid support to remain isolated or otherwiseconcentrated in the sample processing chamber or which cause it to beresuspended in the wash reagent. Resuspending the solid support in thewash reagent may be accompanied by mixing, such as by agitating thereceptacle, a turbulent movement of wash reagent from the wash reagentchamber into the sample processing chamber, or the use of pressingforces to mix the contents of the sample processing chamber. After anappropriate dwell time, the solid support can again be isolated orotherwise concentrated and the wash reagent moved from the sampleprocessing chamber into a waste chamber. This process can be repeated asneeded.

The instrument may include elements for controlling the thermalconditions of one or more chambers or for providing a uniformtemperature within the instrument. Factors to be considered in selectingthermal control elements for use in performing a particular processinclude determining the desired temperature range, the rate of change oftemperature, the accuracy, precision and stability of temperature,whether zonal heating and/or cooling or a uniform temperature isrequired, and the effect of external conditions, as well asheat-producing components of the instrument (e.g., motors), ontemperature control capabilities. The heating and/or cooling of thechambers or subsets of chambers may be accomplished with thermal controlelements that use, for example, electrical changes, radiation,microwaves, sonication, convection, conduction, forced air, chemicalreactions (e.g., exothermic and endothermic reactions), biologicalactivities (e.g., heat-generating growth), circulating fluids (e.g.,heated water or freon), and the like to alter the thermal conditions ofa chamber or chambers. Alternatively, the instrument may be placed in atemperature-controlled environment, such as an incubator orrefrigerator, to maintain a uniform temperature.

A preferred instrument includes thermal conducting plates, such ascopper or aluminum plates, that align with a collection of chambers, ora particular chamber or region of a chamber, when the receptacle isproperly loaded in the instrument. The temperature of each plate can becontrolled by, for example, the use of a thermoelectric device.Depending on the direction of current flow in a thermoelectric device,the junction of dissimilar conductors in the thermoelectric device willeither absorb or release heat. Thus, thermoelectric devices can be usedfor the heating and/or cooling of chambers or regions of chambers. Otheradvantages of thermoelectric devices include their size, the absence ofany moving parts or vibration, rapid temperature changes, precisiontemperature control, and no CFCs or moving fluids.

In practice, the thermal conducting plates are positioned in theinstrument so that they will be in the general proximity of, andpreferably contacting, the chambers or regions of chambers to be heatedor cooled in a properly loaded receptacle. The plates are separated fromeach other using a non-conductive material, such as Ultem® polyimdethermoplastic resin or Delrin® acetyl resin. Using the thermoelectricdevices, heat is transferred by conduction, convection or radiation.

In an alternative embodiment, all or a portion of the thermal controlelements may be associated with actuators which provide localizedheating or cooling of chambers.

The instrument preferably includes at least one detector for detecting asignal or other physical, chemical, biochemical or biological event. Thedetector may be used to detect whether an analyte or multiple analytesare present in a sample material, or present in an altered state. Thedetector, in cooperation with a microprocessor, may also provideinformation about the quantity of an analyte or analytes present in asample material. Detectors contemplated by the present invention includefluorometers, luminometers, spectrophotometers, infrared detectors andcharged-coupled devices. Each of these types of detectors, or signalreceiving components associated with these detectors, can be positionedadjacent a detection chamber for detecting a wide variety of signaltypes. A detector may be mounted on a movable platform so that thedetector can be positioned adjacent different chambers of a stationaryreceptacle. Alternatively, multiple types of detectors may be movablymounted on a platform to facilitate different detection methods fordifferent processes. The instrument may also include multiple detectorsof the same or different types for detecting signals emitted fromdifferent chambers simultaneously. Fiber optics may also be used tocollect signals from different locations and transmit this informationto a detector or detectors at stationary sites removed from thereceptacle. Also contemplated is a fiber optic arrangement incombination with a movable detector. Other possible detectors might beused to detect, for example, radioactive, magnetic or electronic labels,Raman scattering, Surface Plasmon Resonance, gas, turbidity, or a mass,density, temperature, electronic or color change.

Uses of the Receptacles

The receptacles of the present invention can be used, alone or incombination with a cooperating instrument, to perform a variety ofprocesses. Such processes may include, for example, separating orisolating an analyte of interest from other components of a sample,exposing a sample or component of a sample to reagents and conditionsneeded to analyze the sample, and/or performing a chemical, biochemicalor biological reaction which effects a detectable change, such as achange in composition, sequence, volume, quantity, mass, conductivity,turbidity, color, temperature or the like. As discussed above, thereceptacles are particularly suited for use in applications requiring orbenefiting from actions that are performed non-sequentially. These typesof applications include, but are not limited to, complex tests or assaysinvolving detectable binding interactions such as antigen-antibody,nucleic acid-nucleic acid, and receptor-ligand interactions.

Nucleic acid based assays that can be performed in the receptacles ofthe present invention may rely upon direct detection of a target nucleicacid or detection of an amplification product indicative of the presenceof the target nucleic acid in a sample. Direct detection requires thatthere be a sufficient quantity of the target nucleic acid in a sample tosensitively determine the presence of a target sequence associated with,for example, gene expression, a chromosomal abnormality or a pathogenicorganism. Because of the large cellular quantities of ribosomal RNA(rRNA) in non-viral organisms, and the sequence conservation thatenables phylogenetically coherent groupings of organisms to bedistinguished from each other, rRNA is an ideal target for a directdetection assay designed to determine the presence of a pathogenicorganism (e.g., bacterium, fungus or yeast). See, e.g., Kohne, “Methodfor Detecting, Identifying, and Quantitating Organisms and Viruses,”U.S. Pat. No. 5,288,611; and Hogan et al., “Nucleic Acid Probes forDetection and/or Quantitation of Non-Viral Organisms,” U.S. Pat. No.5,840,488. Regardless of the type of nucleic acid being targeted, thesensitivity of a direct detection assay can be improved by a signalamplification procedure in which a probe or probe complex binding to atarget nucleic acid has multiple labels for detection, therebyincreasing the signal of an assay without affecting the amount of targetin the sample. See, e.g., Hogan et al., “Branched Nucleic Acids,” U.S.Pat. No. 5,424,413; and Urdea et al., “Nucleic Acid Multimers andAmplified Nucleic Acid Hybridization Assays Using the Same,” U.S. Pat.No. 5,124,246. Another form of amplification that does not requireincreasing the copy number of a target nucleic acid sequence is probeamplification, which includes procedures such as the Ligase ChainReaction (LCR). LCR relies upon repeated cycles of probe hybridizationand ligation to generate multiple copies of a nucleic acid sequence.See, e.g., Birkenmeyer et al., “Amplification of Target Nucleic AcidUsing Gap Filling Ligase Chain Reaction,” U.S. Pat. No. 5,427,930. Othercontemplated signal amplification procedures include those utilizingThird Wave Technology's Invader® chemistry. See, e.g., Kwiatkowski etal., “Clinical, Genetic, and Pharmacogenetic Applications of the InvaderAssay,” Mol. Diagn. (1999) 4(4):353-64.

Target nucleic acid amplification involves the use of amplificationoligonucleotides (e.g., primers) and polymerases to enzymaticallysynthesize nucleic acid amplification products (copies) containing asequence that is either complementary or homologous to the templatenucleic acid sequence being amplified. The amplification products may beeither extension products or transcripts generated in atranscription-based amplification procedure. The amplificationoligonucleotides may be provided to a reaction mixture free in solutionor one or more of the amplification oligonucleotides may be immobilizedon a solid support, including the inner surface of a chamber or chamberswithin a receptacle. See, e.g., Adams et al., “Method for PerformingAmplification of Nucleic Acid with Two Primers Bound to a Single SolidSupport,” U.S. Pat. No. 5,641,658; and Browne, “Nucleic AcidAmplification and Detection Method,” U.S. Patent Application PublicationNo. US 2005-0287591 A1. Examples of nucleic acid amplificationprocedures practiced in the art include the polymerase chain reaction(PCR), strand displacement amplification (SDA), helicase dependentamplification (HDA), loop-mediated isothermal amplification (LAMP), anda variety of transcription-based amplification procedures, includingtranscription-mediated amplification (TMA), nucleic acid sequence basedamplification (NASBA), and self-sustained sequence replication (3SR).See, e.g., Mullis, “Process for Amplifying, Detecting, and/or CloningNucleic Acid Sequences,” U.S. Pat. No. 4,683,195; Walker, “StrandDisplacement Amplification,” U.S. Pat. No. 5,455,166; Kong et al.,“Helicase Dependent Amplification of Nucleic Acids,” U.S. Pat. No.7,282,328, Notomi et al., “Process for Synthesizing Nucleic Acid,” U.S.Pat. No. 6,410,278; Kacian et al., “Nucleic Acid Sequence AmplificationMethods,” U.S. Pat. No. 5,399,491; Becker et al., Single-Primer NucleicAcid Amplification Methods,” U.S. Pat. No. 7,374,885; Malek et al.,“Enhanced Nucleic Acid Amplification Process,” U.S. Pat. No. 5,130,238;and Lizardi et al. (1988) BioTechnology 6:1197. With some procedures,the formation of detectable amplification products depends on an initialantibody/antigen interaction. See, e.g., Cashman, “Blocked-PolymerasePolynucleotide Immunoassay Method and Kit,” U.S. Pat. No. 5,849,478.Nucleic acid amplification is especially beneficial when the amount ofanalyte (e.g., targeted nucleic acid, antigen or antibody) present in asample is very low. By amplifying a target sequence associated with theanalyte and detecting the synthesized amplification product, thesensitivity of an assay can be vastly improved, since less analyte isneeded at the beginning of the assay to ensure detection of the analyte.

The conditions of a target nucleic acid amplification reaction may besubstantially isothermal or they may require periodic temperaturechanges, as with PCR thermal cycling. The instrument described supra mayprovide a constant or ambient temperature or it may be modified andprogrammed to fluctuate the overall temperature within the instrumentor, alternatively, particular zones of the instrument which affectspecific chambers of a receptacle. Target nucleic acid amplificationreactions may be either “real-time” or “end-point” assays. A compact,lightweight, multi-channel fluorometer that is particularly suited foruse in performing real-time assays in an instrument of the presentinvention is described below. Real-time amplification assays involveperiodically determining the amount of targeted amplification productsas the amplification reaction is taking place, thereby making it easierto provide quantitative information about an analyte (e.g. targetnucleic acid) present in a sample, whereas end-point amplificationsdetermine the amount of targeted amplification products after theamplification reaction has occurred, generally making them more usefulfor providing qualitative information about an analyte present in asample. Algorithms for calculating the quantity of target nucleic acidor other analyte originally present in a sample based on signalinformation collected during or at the completion of an amplificationreaction include those disclosed by Wittwer et al., “PCR Method forNucleic Acid Quantification Utilizing Second or Third Order RateConstants,” U.S. Pat. No. 6,232,079; Sagner et al., “Method for theEfficiency-Corrected Real-Time Quantification of Nucleic Acids,” U.S.Pat. No. 6,691,041; McMillan et al., “Methods for Quantitative Analysisof a Nucleic Acid Amplification Reaction,” U.S. Pat. No. 6,911,327;Light et al., “Method for Determining the Amount of an Analyte in aSample,” U.S. Patent Application Publication No. US 2006-0276972 A1;Chismar et al., “Method and Algorithm for Quantifying Polynucleotides,”U.S. Patent Application Publication No. US 2006-0292619 A1; and Ryder etal., “Methods for Determining Pre-Amplification Levels of a Nucleic AcidTarget Sequence from Post-Amplification Levels of Product,” U.S. Pat.No. 5,710,029. Also, to confirm that the amplification conditions andreagents were appropriate for amplification, it is generally desirableto provide an internal control sequence at the start of a nucleic acidamplification reaction. See, e.g., Wang et al., “Quantitation of NucleicAcids Using the Polymerase Chain Reaction,” U.S. Pat. No. 5,476,774.

Detection of a target nucleic acid may be in situ or in vitro. See,e.g., Gray et al., “Methods for Chromosome-Specific Staining,” U.S. Pat.No. 5,447,841. For in vitro assays, it may be necessary to lyse orpermeabilize cells to first release the targeted nucleic acid and makeit available for hybridization with a detection probe. See, e.g., Clarket al., “Method for Extracting Nucleic Acids from a Wide Range ofOrganisms,” U.S. Pat. No. 5,786,208. If the cells are lysed, thecontents of the resulting lysate may include, in addition to nucleicacids, organelles, proteins (including enzymes such as proteases andnucleases), carbohydrates, and lipids, which may necessitate furtherpurification of the nucleic acids. Additionally, for pathogenicorganisms, chemical or thermal inactivation of the organisms may bedesirable. The cells may be lysed or permeabilized prior to loadingsample into a receptacle of the present invention, or a sample or otherchamber of the receptacle may be pre-loaded with an agent for performingthis function. Cells may be lysed or permeabilized by a variety of meanswell known to those skilled in the art, including by chemical,mechanical (e.g., sonication) and/or thermal means. One preferred lyticagent in described in the Examples section below.

Released nucleic acids can be isolated or separated in the receptaclesfrom other sample components that may act as inhibitors which interferewith the detection and/or amplification of a target sequence. Thepresence of potentially interfering components will vary depending onthe sample type and may include components of the cell lysate, such asnucleases that can digest the released and targeted nucleic acids. Someunwanted sample components may be separated from the target nucleic acidthrough precipitation or solid phase capture by providing to a chambersuch materials as filters, beads, fibers, membranes, glass wool, filterpaper, polymers or gels. Suitable filters include glass, fiberglass,nylon, nylon derivatives, cellulose, cellulose derivatives, and otherpolymers. Alternatively, a solid phase material may be used to capturesample components for lysing, such as cells, spores or microorganisms,where the components may be captured by physical retention (e.g., sizeexclusion, affinity retention, or chemical selection).

Various solid phase methods for capturing nucleic acids are known in theart and can be readily adapted for use in the receptacles of the presentinvention. These methods may be specific or non-specific for thetargeted nucleic acid. One such method is Solid Phase ReversibleImmobilization, which is based on the selective immobilization ofnucleic acids onto magnetic microparticles having carboxyl group-coatedsurfaces. See Hawkins, “DNA Purification and Isolation Using MagneticParticles,” U.S. Pat. No. 5,705,628. In another method, magneticparticles having poly(dT) sequences derivatized thereon bind to captureprobes having 5′ poly(dA) tails and 3′ target binding sequences. See,e.g., Weisburg et al., “Two-Step Hybridization and Capture of aPolynucleotide,” U.S. Pat. No. 6,534,273. Yet another commonly usedmethod binds nucleic acids to silica or glass particles in the presenceof guanidinium thiocyanate, which is a known agent for lysing cells andinactivating nucleases. Still another approach is based on theChargeSwitch® Technology, which is a magnetic bead-based technology thatprovides a switchable surface that is charge dependent on thesurrounding buffer pH to facilitate nucleic acid purification(Invitrogen Corporation, Carlsbad, Calif.; Cat. No. CS12000). In low pHconditions, the ChargeSwitch® Magnetic Beads have a positive charge thatbinds the negatively charged nucleic acid backbone. Proteins and othercontaminants that are not bound can be washed away. By raising the pH to8.5, the charge on the surface is neutralized and the bound nucleicacids are eluted.

In another approach, capture probes that are capable of binding to thetargeted nucleic acid (or to intermediate oligonucleotides that alsobind to the targeted nucleic acids) are covalently or non-covalentlyattached to an inner surface of a designated sample processing chamberduring manufacture of the receptacle. Attachment chemistries are wellknown to skilled artisans and include amine and carboxylic acid modifiedsurfaces for covalent attachment of oligonucleotides and biotin-labeledoligonucleotides and avidin- or streptavidin-coated surfaces fornon-covalent attachments. With this approach, targeted nucleic acidsintroduced into the sample processing chamber can be immobilized on thesurface of the chamber and liquid and other unbound materials can beremoved without having to immobilize or trap particles or beads.Following separation from other materials of the sample, the targetednucleic acids may remain immobilized on the surface for amplificationand detection or they may be first eluted from the capture probes.Alternatively, the capture probes could be immobilized on a porous solidsupport, such as a sponge, that is located in the sample processingchamber.

Capture probes suitable for use in the present invention may be specificor non-specific for the targeted nucleic acids. A specific capture probewill include a target binding region that is selected to bind to atarget nucleic acid under a predetermined set of conditions and not tonon-target nucleic acids which may be present in a sample. Anon-specific capture probe does not discriminate between target andnon-target nucleic acids under the conditions of use. Wobble captureprobes are an example of a non-specific capture probe and may include atleast one random or non-random poly(K) sequence, where “K” can representa guanine, thymine or uracil base. See Becker et al., “Methods ofNonspecific Target Capture of Nucleic Acids,” U.S. patent applicationSer. No. 11/832,367, which enjoys common ownership herewith. In additionto hydrogen bonding with cytosine, its pyrimidine complement, guaninewill also hydrogen bond with thymine and uracil. Each “K” may alsorepresent a degenerate nucleoside such as inosine or nebularine, auniversal base such as 3-nitropyrrole, 5-nitroindole or 4-methylindone,or a pyrimidine or purine base analog such as dP or dK. The poly(K)sequence of a wobble capture probe is of sufficient length tonon-specifically bind the target nucleic acid, and is preferably 6 to 25bases in length.

Formats for detecting a target nucleic acid or related amplificationproduct can be divided into two basic categories: heterogeneous andhomogeneous. Both of these detection formats can be adapted for use inthe receptacles of the present invention. Heterogeneous assays include astep to separate bound from unbound probe, while no such physicalseparation step is used in homogeneous assays. Numerous heterogeneousand homogeneous detection methods are known in the art. See, e.g., Jung,P. et al. 1997. “Labels and Detection Formats in Amplification Assays.”In Nucleic Acid Amplification Technologies, eds. Lee, H. et al.,135-150. Natick, Mass.: BioTechnique Books.

Assay methods utilizing a physical separation step include methodsemploying a solid-phase matrix, such as glass, minerals or polymericmaterials, in the separation process. The separation may involvepreferentially binding the probe:analyte complex to the solid phasematrix, while allowing the unassociated probe molecules to remain in aliquid phase. Such binding may be non-specific, as, for example, in thecase of hydroxyapatite, or specific, for example, throughsequence-specific interaction of the target nucleic acid with a captureprobe that is directly or indirectly immobilized on the solid support.In any such case, the amount of probe remaining bound to the solid phasesupport after a washing step is proportional to the amount of analyte inthe sample.

Alternatively, the assay may involve preferentially binding theunhybridized probe while probe:analyte complexes remain in the liquidphase. In this case the amount of probe in the liquid phase after awashing step is proportional to the amount of analyte in the originalsample. When the probe is a nucleic acid or oligonucleotide, the solidsupport can include, without limitation, an adsorbent such ashydroxyapatite, a polycationic moiety, a hydrophobic or “reverse phase”material, an ion-exchange matrix, such as DEAE, a gel filtration matrix,or a combination of one or more of these solid phase materials. Thesolid support may contain one or more oligonucleotides, or otherspecific binding moiety, to capture, directly or indirectly, probe,target, or both. In the case of media, such as gel filtration,polyacrylamide gel or agarose gel, the separation is not due to bindingof the oligonucleotide but is caused by molecular sieving of differentlysized or shaped molecules. In the latter two cases, separation may bedriven electrophoretically by application of an electrical currentthrough the gel causing the differential migration through the gel ofnucleic acids of different sizes or shapes, such as double-stranded andsingle-stranded nucleic acids.

A heterogeneous assay method may also involve binding the probe to asolid-phase matrix prior to addition of a sample suspected of containingthe analyte of interest. The sample can be contacted with the labelunder conditions that would cause the desired nucleic acid to belabeled, if present in the sample mixture. The solid phase matrix may bederivatized or activated so that a covalent bond is formed between theprobe and the matrix. Alternatively, the probe may be bound to thematrix through strong non-covalent interactions, including, withoutlimitation, the following interactions: ionic, hydrophobic,reverse-phase, immunobinding, chelating, and enzyme-substrate. After thematrix-bound probe is exposed to the labeled nucleic acid underconditions allowing the formation of a hybrid, the separation step isaccomplished by washing the solid-phase matrix free of any unbound,labeled analyte. Conversely, the analyte can be bound to the solid phasematrix and contacted with labeled probe, with the excess free probewashed from the matrix before detection of the label.

As noted above, homogenous assays take place in solution, without asolid phase separation step, and commonly exploit chemical differencesbetween free probe and probe:analyte complexes. An example of an assaysystem that can be used in a homogenous or heterogeneous format is thehybridization protection assay (HPA). See Arnold et al., “HomogenousProtection Assay,” U.S. Pat. No. 5,283,174. In HPA, a probe is linked toa chemiluminescent moiety, contacted with a sample and then subjected toselective chemical degradation or a detectable change in stability underconditions which alter the chemiluminescent reagent linked tounhybridized probe without altering the chemiluminescent reagent linkedto a probe:analyte complex. Subsequent initiation of a chemiluminescentreaction causes the hybrid-associated label to emit light.

Other homogeneous assays rely upon a physical alteration to a detectionprobe or amplification primer to provide a detectable signal changeindicative of the presence of a target nucleic acid. Probes and primerscapable of undergoing detectable physical alterations include, but arenot limited to, self-hybridizing probes, such as molecular beacons ormolecular torches, bi-molecular probes, TaqMan® probes that arecommercially available from Applied Biosystems, Lux™ primers that arecommercially available from Invitrogen Corporation, and signal primers.See, e.g., Tyagi et al. (1996) Nature Biotechnology 14(3):303-308;Becker et al., “Molecular Torches,” U.S. Pat. No. 6,849,412; Morrison,“Competitive Homogeneous Assay,” U.S. Pat. No. 5,928,862; Tapp et al.(2000) BioTechniques 28(4):732-738; Nazarenko (2006) Methods Mol. Biol.335:95-114; and Nazarenko (1997) Nucleic Acids Res. 25(12):2516-2521.Each of these probes and primers relies upon a conformational change inthe probe or primer upon hybridization to a target nucleic acid torender a detectable change in an associated reporter moiety (e.g.,fluorescent molecule). Prior to hybridization, signal from the reportermoiety may be altered by an associated quencher moiety which, in thecase of Lux™ primers, is a guinine located near the 3′ end of the primersequence.

Particularly preferred detection probes for use in real-timeamplification reactions are self-hybridizing probes that emitdifferentially detectable signals, depending on whether the probesremain self-hybridized or bind to amplification products. The probes maybe provided to a reaction mixture free in solution or immobilized onsolid supports. See, e.g., Cass et al., “Immobilized Nucleic AcidHybridization Reagent and Method,” U.S. Pat. No. 6,312,906.Advantageously, they may also be provided to a reaction mixture before,after or at the time an amplification reaction has been initiated. Ifthe probes are provided on a solid support, then the solid support mayadditionally include one or more immobilized amplificationoligonucleotides for amplifying a target nucleic acid sequence.Preferred self-hybridizing probes include molecular beacons andmolecular torches.

Molecular beacons comprise nucleic acid molecules or analogs thereofhaving a target complementary sequence, an affinity pair (or nucleicacid or nucleic acid analog arms or stems) holding the probe in a closedconformation in the absence of a target nucleic acid sequence, and alabel pair that interacts when the probe is in a closed conformation.See Tyagi et al., “Detectably Labeled Dual Conformation OligonucleotideProbes, Assays and Kits,” U.S. Pat. No. 5,925,517. Hybridization of thetarget nucleic acid and the target complementary sequence separates themembers of the affinity pair, thereby shifting the probe to an openconformation. The shift to the open conformation is detectable due toreduced interaction of the label pair, which may be, for example, afluorophore and a quencher (e.g., DABCYL and EDANS).

Molecular torches have distinct regions of self-complementarity,described as the “target binding” and “target closing” domains. Thesedomains are linked by a joining region and are sufficientlycomplementary to hybridize to each other under predeterminedhybridization assay conditions. When exposed to denaturing conditions,the complementary regions melt, leaving the target binding domainavailable for hybridization to a target sequence when the predeterminedhybridization assay conditions are restored. And when exposed to stranddisplacement conditions, a portion of the target sequence binds to thetarget binding domain, thereby displacing the target closing domain fromthe target binding domain. Molecular torches are designed so that thetarget binding domain favors hybridization to the target sequence overthe target closing domain. The target binding domain and the targetclosing domain of a molecular torch include interacting labelspositioned so that a different signal is produced when the moleculartorch is self-hybridized than when it is hybridized to a target nucleicacid, thereby permitting detection of probe:target complexes in a testsample in the presence of unhybridized probe having a viable label orlabels associated therewith.

Different types of interacting moieties can be used to determine whethera probe has undergone a conformational change. For example, theinteracting moieties may be a luminescent/quencher pair, aluminescent/adduct pair, a Forrester energy transfer pair or a dyedimer. More than one type of label may be present on a particularmolecule.

A luminescent/quencher pair is made up of one or more luminescentmoieties, such as chemiluminescent or fluorescent moieties, and one ormore quenchers. Preferably, a fluorescent/quencher pair is used todetect a probe that has undergone a conformational change. A fluorescentmoiety absorbs light of a particular wavelength, or wavelength range,and emits light with a particular emission wavelength, or wavelengthrange. A quencher moiety dampens, partially or completely, signalemitted from an excited fluorescent moiety. Quencher moieties can dampensignal production from different fluorophores. For example, DABCLY([4-(4′-dimethylaminophenylazo) benzoic acid]) can quench about 95% ofthe signal produced from EDANS(5-(2′-aminoethyl)aminoaphthaline-1-sulfonic acid), rhodamine andfluorescein.

Different numbers and types of fluorescent and quencher moieties can beused. For example, multiple fluorescent moieties can be used to increasesignal production from an opened molecular beacon or torch, and multiplequencher moieties can be used to help ensure that, in the absence of atarget sequence, an excited fluorescent molecule produces little or nosignal. Examples of fluorophores include acridine, fluorescein,sulforhodamine 101, rhodamine, EDANS, Texas Red, Eosine, Bodipy andlucifer yellow. See, e.g., Tyagi et al. (1998) Nature Biotechnology16:49-53. Examples of quenchers include DABCYL, Thallium, Cesium, andp-xylene-bis-pyridinium bromide.

A luminescent/adduct pair is made up of one or more luminescent moietiesand one or more molecules able to form an adduct with the luminescentmolecule(s) and, thereby, diminish signal production from theluminescent molecule(s). The use of adduct formation to alter signalsfrom a luminescent molecule using ligands free in solution is disclosedby Becker et al., “Adduct Protection Assay,” U.S. Pat. No. 5,731,148.

Förrester energy transfer pairs are made up of two moieties, where theemission spectra of a first moiety overlaps with the excitation spectraof a second moiety. The first moiety can be excited and emissioncharacteristic of the second moiety can be measured to determine if themoieties are interacting. Examples of Förrester energy transfer pairsinclude pairs involving fluorescein and rhodamine;nitrobenz-2-oxa-1,3-diazole and rhodamine; fluorescein andtetramethylrhodamine; fluorescein and fluorescein; IAEDANS andfluorescein; and BODIPYFL and BIODIPYFL.

Dye dimers comprise two dyes that interact upon the formation of a dimerto produce a different signal than when the dyes are not in a dimerconformation. See, e.g., Packard et al. (1996) Proc. Natl. Acad. Sci.USA 93:11640-11645.

While homogeneous assays are generally preferred, essentially anylabeling and detection system that can be used for monitoring specificnucleic acid hybridization can be used in conjunction with thereceptacles of the present invention. Included among the collection ofuseful labels are radiolabels, intercalating dyes, enzymes, haptens,linked oligonucleotides, chemiluminescent molecules and redox-activemoieties that are amenable to electronic detection methods. Preferredchemiluminescent molecules include acridinium esters of the typedisclosed by Arnold et al. in “Homogeneous Protection Assay,” U.S. Pat.No. 5,283,174 for use in connection with hybridization protection assays(HPA), and of the type disclosed by Woodhead et al. in “Detecting orQuantifying Multiple Analytes Using Labeling Techniques,” U.S. Pat. No.5,656,207 for use in connection with assays that quantify multipletargets in a single reaction. Preferred electronic labeling anddetection approaches are disclosed by Meade et al., “Nucleic AcidMediated Electron Transfer,” U.S. Pat. No. 5,591,578, and Meade,“Detection of Analytes Using Reorganization Energy,” U.S. Pat. No.6,013,170. Redox active moieties useful as labels include transitionmetals such as Cd, Mg, Cu, Co, Pd, Zn, Fe and Ru.

Synthetic techniques and methods of bonding reporter moieties to nucleicacids and detecting reporter moieties are well known in the art. See,e.g., J. SAMBROOK ET AL., MOLECULAR CLONING: A LABORATORY MANUAL,Chapter 10 (2d ed. 1989); Becker et al., U.S. Pat. No. 6,361,945; Tyagiet al., U.S. Pat. No. 5,925,517, Tyagi et al., “Nucleic Acid DetectionProbes Having Non-FRET Fluorescence Quenching and Kits and AssaysIncluding Such Probes,” U.S. Pat. No. 6,150,097; Nelson et al., U.S.Pat. No. 5,658,737; Woodhead et al., U.S. Pat. No. 5,656,207; Hogan etal., “Nucleic Acid Probes for Detection and/or Quantitation of Non-ViralOrganisms,” U.S. Pat. No. 5,547,842; Arnold et al., U.S. Pat. No.5,283,174; Kourilsky et al., “Method of Detecting and Characterizing aNucleic Acid or Reactant for the Application of this Method,” U.S. Pat.No. 4,581,333; and Becker et al., U.S. Pat. No. 5,731,148.

Process materials may be provided to the chambers of a receptacle in adried or liquid form. Providing process materials in a dried form can beespecially beneficial where the process materials are, in their liquidform, unstable, biologically or chemically active, temperature sensitiveor chemically reactive with each other. Drying inhibits the activity ofmicroorganisms and enzymes and can improve the shelf-life and storageconditions of process materials (room temperature as opposed to coldstorage). Dried process materials, in addition to their reactivecomponents, may include a cryoprotectant (e.g., disaccharides such assucrose, maltose, lactose or trehalose) to help preserve the biologicalactivity of a material as it is being frozen, dried and/or reconstitutedand a stabilizing agent (e.g., various sugars including sucrose andtrehalose, sugar alcohols and proteins) to prevent or delay the loss ofa material s biological activity over time. The suitability of any givencryoprotectant or stabilizing agent will depend on the nature of thematerial being dried. After drying, the process materials should besealed to prevent reabsorption of moisture. Methods and devices fordrying process materials are well known in the art and includelyophilization or freeze-drying. See, e.g., Price et al., “PelletizedPregnancy Test Reagents,” U.S. Pat. No. 3,862,302; Temple et al,“Process for Freezing or Chilling,” U.S. Pat. No. 4,655,047; Milankov etal., “Cryogenic Apparatus,” U.S. Pat. No. 4,982,577; Shen et al.,“Stabilized Enzyme Compositions for Nucleic Acid Amplification,” U.S.Pat. No. 5,834,254; Buhl et al., “Dried Chemical Compositions,” U.S.Pat. No. 6,251,684; and McMillan, “Universal and Target Specific ReagentBeads for Nucleic Acid Amplification,” U.S. Patent ApplicationPublication No. US 2006-0068398 A1.

Illustrative Embodiments

An exemplary embodiment of a multi-chambered receptacle embodyingaspects of the invention is designated by reference number 10 in FIG.1A. In this embodiment, the receptacle 10 is a generally planar vesselhaving flexible top and bottom sheets formed from thin flexiblematerials, such as foils and/or plastics, and an upper edge 12 and alower edge 14 that indicate the preferred orientation of the receptacleduring use and define an upper direction and a lower direction. Theexemplary receptacle 10 has dimensions of about 7.5 inches by about 3.2inches and is less than about ¼ inch thick (when filled with sample andprocess materials), but may be of any dimensions suitable for manualmanipulation or for use with an automated system, such as the onedescribed herein. In general, the dimensions of the receptacle mustaccommodate the substances needed to conduct a process or set ofprocesses. Persons of skill will recognize that a wide variety of sizes,conformations, shapes, and the like are compatible with variousprocesses and are contemplated for use.

The receptacle may include one or more attachment or alignment holes 74that register with structures, such as hooks or pins, of an automatedinstrument for mounting and/or alignment of the receptacle with respectto the instrument. One or more labels for identification of the sample,patient or any other information of interest, including test conditionsand parameters optionally may be provided on a receptacle surface orembedded in material used to construct the receptacle. Such labels caninclude indicia that are human readable, machine readable (e.g.,barcodes), Optical Character Recognition (OCR), Radio FrequencyIdentification (RFID), or some combination thereof. Referring to FIG.1A, receptacle 10 comprises a number of chambers that form part of anintegrated system, where the chambers collectively define a plurality ofnon-linear pathways punctuated with selectively openable connections.Inlet port 52 and neck portion 51 serve as a channel for receiving asample or other material for processing, testing or subjecting toreactants and may have any suitable configuration for admission of thesample or other material. Sample material may be transferred to thereceptacle 10 by any suitable means, for example, by using a syringewith needle that punctures the inlet port 52. The inlet port 52alternatively may comprise the female portion of a Luer connection forinsertion of a syringe or other container having a male Luer connectionor an unsealed opening in the top of the receptacle through which thesample material may be poured, pipetted or otherwise inserted. The inletport 52 is preferably located at or near the upper edge 12 of thereceptacle 10 to reduce the potential for spillage of sample materialupon its transfer or prior to placement of the receptacle into anautomated instrument. However, the inlet port 52 may be located at anyedge of the receptacle 10 or located more centrally, as is convenient,for example as a slot or other opening, which optionally is reversiblysealed. For example, the inlet port may be closed by heat sealing theopposed sheets of the receptacle 10 after admission of sample material.

The integrated chamber system of the illustrated receptacle 10 includeseleven chambers C16, C18, C20, C22, C24, C26, C28, C30, C32, C34 andC36. The chambers are generally enclosed compartments that may beconnected (selectively, temporarily, or permanently) with one or moreadjacent chambers so as to permit substances to flow between at least aportion of the adjacent chambers, as well as between various chambers ofthe integrated system. Each chamber may contain a substance used toperform a process within the receptacle 10 such as, for example, samplematerial, sample processing reagents for preparing a sample material forfurther analysis, reactants, solvents, diluents, wash reagents and thelike. Furthermore, chambers may function, either through manualmanipulation or in cooperation with various elements of an instrument,as the locus for performing one or more process steps, such as ananalyte purification procedure, mixing, heating/cooling, detection of asignal or visual characteristic (e.g. color change), waste storage andremoval, etc. Some chambers may be pre-loaded with substances, e.g.,sample, reaction reagents, buffers, etc., when the receptacle 10 isplaced in an instrument and other chambers may be initially empty, butone or more substances may be moved into or through the initially-emptychamber when performing a process. Some chambers may not be used at all,depending on the requirements of the particular process being performedwithin the receptacle 10.

In an exemplary use of the receptacle 10, the chambers can be filledwith substances needed to perform a binding reaction, such as animmunoassay or a nucleic acid-based reaction. In such an application ofthe receptacle 10, chamber C16 may be loaded with sample material,chamber C18 may be loaded with a sample processing reagent for bindingand immobilizing an analyte present in the sample material on a solidsupport, chamber C26 may function as a sample processing chamber forseparating the immobilized analyte from other components of the samplematerial, chamber C22 may be loaded with a dried, first processmaterial, chamber C20 may be loaded with a reagent for reconstitutingthe first process material, chamber C28 may be loaded with a dried,second process material, chamber C30 may be loaded with a reagent forreconstituting the second process material, chamber C34 may be loadedwith a wash reagent, and chamber C36 may function as a waste chamberinto which waste substances are moved when performing a process and inwhich those waste substances are stored in relative isolation from otheraspects of the process. In addition to containing the second processmaterial, chamber C28 may also function as a detection chamber fordetecting a signal or change in a reaction mixture that is indicative ofthe presence of the analyte in the sample material. In an alternativeimplementation of the receptacle 10, chamber C32 could contain a reagentfor reconstituting a dried, second process material contained in chamberC30, which then could be used to reconstitute a dried, third processmaterial contained in chamber C28. Alternatively, a dried, secondprocess material could be loaded in chamber C30 and a reagent forreconstituting the second process material could be loaded in chamberC32, where reconstituted forms of the first and second process materialscould be combined with the separated analyte in chamber C28.

Other non-limiting uses of the receptacle 10 will be described in theExamples section of the disclosure.

In the illustrated embodiment, chamber C34 is specially designed tocontain a wash reagent and includes an upper portion 38, a lower neck40, a vertical section 42, and a lateral section 44 extending towardchamber C26.

Also, in the illustrated embodiment of the receptacle 10, chamber C36 isadvantageously configured to function as a waste chamber for receivingwaste materials from chamber C26 and includes an initial vertical inlet48 extending from chamber C26, an upper neck 46, and a collection region50. Vertical inlet 48 is positioned above chamber C26 and is connectedto chamber C26 by means of portal 70 positioned at the top of chamberC26. When an analyte separation procedure, such as a magnetic separationprocedure, is performed in chamber C26, bubbles may be formed in ormoved into chamber C26 by, for example, a detergent present in thereaction mixture (e.g., a detergent-based lytic agent provided to thesample or detergent present in a sample processing reagent).

As illustrated in FIG. 1A, the chambers of the receptacle 10 areinterconnected as follows: chamber C18 is connected to chamber C16 byportal 54; chamber C16 is connected to chamber C26 by portal 62; chamberC20 is connected to chamber C22 by portal 56; chamber C22 is connectedto chamber C26 by portal 58; chamber C24 is connected to chamber C26 byportal 60; chamber C32 is connected to chamber C30 by portal 68; chamberC30 is connected to chamber C28 by portal 66; chamber C28 is connectedto chamber C26 by portal 64; chamber C34 is separately connected tochamber C26 by portal 72; and, as noted above, chamber C26 is connectedto chamber C36 by portal 70. In some embodiments, one or more of portals54, 56, 58, 60, 62, 64, 66, 68, 70, and 72 is temporarily closed toprevent fluid flow therethrough by an openable seal, such as a heat sealthat peels open when pressure is applied to a connected chamber.

In the embodiment of FIG. 1A, chamber C16 of the receptacle 10 isconfigured to hold a suitable sample volume. Generally, the sample willbe a fluid or fluidized sample, such as a fluid sample taken from ahuman or other animal, which may include blood or a blood product (i.e.,plasma or serum), cerebral spinal fluid, a conjunctiva specimen, arespiratory specimen, a nasopharyngeal specimen, or a genitourinarytract specimen, or it may be, for example, an environmental, industrial,food, beverage or water sample. Solid or viscous sample materials (e.g.,food, fecal matter and sputum) will generally need to be at leastpartially solubilized prior to adding the sample material to chamber C16(although it is also possible to solubilize such sample materialsdirectly in the receptacle as well). The sample material may be organicor inorganic, and it may be a material for processing or analysis or areactant in a chemical, biochemical or biological reaction.

The volume capacity of chamber C16 is preferably from about 10 μL toabout 1 mL, more preferably up to about 850 μL, and most preferablyabout 625 μL. This volume capacity is intended to accommodate the totalvolume of substance expected to be placed in chamber C16, which in theexemplary application described herein, includes a volume of samplematerial combined with the sample processing reagent from chamber C18.At the most preferred volume amounts, this would be a 500 μL samplecombined with 125 μL of a sample processing reagent. At the low end ofthe expected volume range there needs to be enough fluid in the chamberC16 to force open the seal at portal 62 upon the application of externalpressure to chamber C16. At the upper end of the expected volume range,the volume placed in chamber C16 cannot be so great that there isstretching of the receptacle and perhaps peeling or rupturing of a wallof the chamber.

Portal 54 connects chamber C18 to chamber C16 and is temporarily closedby a selectively openable seal. Upon application of sufficientcompressive force to chamber C18 by a pressure application mechanism, anexample of which is described below, the seal closing portal 54 isopened and the sample processing reagent is moved from chamber C18 tochamber C16, where it is mixed with the sample material provided tochamber C16. The sample processing reagent preferably includes a bindingagent and a solid support, such as magnetically-responsive particles,for immobilizing the analyte.

The first process material is contained in chamber C22, and thereconstitution reagent for the first process material is contained inchamber C20. In one embodiment, the first process material is anamplification reagent. Dried or solid amplification reagents arepreferred because they are more stable than liquid amplificationreagents. Suitable carriers for the amplification reagent include anychemically inert or compatible material and may optionally include, forexample, diluents, binding agents, lubricants, dissolution aids,preservatives and the like. In one embodiment, amplification reagentsare frozen in a cryogenic fluid to form uniformly sized pellets, whichare then lyophilized for use in unit dose applications. Solid forms ofthe amplification reagents can also be compressed into pellets ortablets, but may be in the form of powders, granules, or any otherconvenient and stable solid form. Dried amplification reagents are alsopreferred because there is less chance of an accidental rupture withdried reagents than with liquid reagents, and solid materials providefor very precise dosing of reagents.

If the first process material is an amplification reagent, then theamplification reagent may contain at least one amplificationoligonucleotide, such as a primer, a promoter-primer, and/or anon-extendable promoter-provider oligonucleotide, nucleosidetriphosphates, and cofactors, such as magnesium ions, in a suitablebuffer. The specific components of the amplification reagent will dependon the amplification procedure to be practiced. An exemplaryamplification reagent for performing a transcription-based amplificationreaction is described in the examples portion of this disclosure.

In some embodiments, chamber C20 is left empty or is omitted altogetherif the first process material is omitted or is provided in a fluid formin chamber C22. Alternatively, chamber C22 could be left empty andliquid loaded in chamber C20.

Upon application of a sufficient compressive force to chamber C20 by apressure application mechanism, such as the one described below, theseal closing portal 56 is opened and the reconstitution reagentcontained in chamber C20 is transferred to the first process materialcontained in chamber C22.

In embodiments in which the reconstitution reagent is not provided(e.g., chamber C20 is empty or omitted in the illustrated receptacle10), the first process material may be a liquid or a solid that ispre-dissolved prior to loading in chamber C22. If the first processmaterial is an amplification reagent, then the amplificationoligonucleotides are preferably present in great excess. Appropriateamounts of these and other reagents can be determined by the skilledartisan and will depend on the assay parameters and the amount and typeof target to be detected.

After the reconstitution reagent is transferred from chamber C20 tochamber C22, a pressure application mechanism applies an externalpressure to chamber C22 that opens the seal closing portal 58 betweenchamber C22 and chamber C26, thereby causing a reconstituted form of thefirst process material to flow from chamber C22 to chamber C26. In someembodiments, the contents of chambers C20 and C22 are mixed, such as bymoving the combined materials between chambers C20 and C22 severaltimes, prior to transferring the reconstituted form of the first processmaterial to chamber C26.

A rinse, if used, follows a wash step and is intended to removesubstances present in the wash reagent that might interfere withprocessing of the analyte. The rinse reagent is contained in chamber C24and preferably comprises an aqueous buffered solution containingdetergent or functionally similar material. The rinse reagent could be areconstituted form of the first process material (e.g., an amplificationreagent without nucleoside triphosphates). Alternatively, the rinsereagent may be a buffer containing no detergents, no anionic detergents,a lower concentration of anionic detergents than the wash reagent, or anonionic detergent to counteract the effect of an anionic detergentpresent in the wash reagent. The volume of rinse reagent contained inchamber C24 in preferred embodiments is from about 150 μL to about 500μL. At the appropriate time, a pressure application mechanism appliespressure on chamber C24 and produces fluid pressure that opens the sealclosing the portal 60 between chamber C24 and chamber C26, allowing therinse reagent to flow from chamber C24 to chamber C26.

Chamber C30 contains a reagent for reconstituting the second processmaterial. In preferred embodiments, the volume of the reconstitutionreagent contained in chamber C30 is from about 20 to about 125 μL, andmore preferably from about 25 to about 100 μL. If the receptacle is usedto perform a nucleic acid-based amplification reaction, then the secondprocess material may contain one or more enzymes, such as polymerasesfor effecting extension and/or transcription of a target sequence and,optionally, a probe that specifically and detectably binds to anamplification product containing the target sequence or its complement.Suitable carriers for solid enzyme and/or probe reagents include anychemically inert or compatible material and may optionally include, forexample, diluents, binding agents, lubricants, dissolution aids,preservatives and the like. The enzyme and/or probe reagents can befrozen in a cryogenic fluid to form uniformly sized pellets, which arethen lyophilized for use in unit dose applications. Solid forms of theenzyme and probe reagents can also be compressed into pellets or tabletswith suitable carriers for ease of handling, but may be in the form of apowder, granules, or any convenient and stable solid form. The two solidcompositions may be formulated as separate pellets or combined as asingle solid form. Furthermore, a dried probe reagent may be loaded intoa chamber, such as chamber C28, in, for example, pellet or granule form,or it may be sprayed, printed or otherwise applied to the walls of achamber.

In reconstituting dried process materials, it was observed that thereconstitution reagents have a tendency to concentrate along theperimeters of the chambers (e.g., heat sealed regions defining thechamber walls), such that dried process materials that are morecentrally located in the chambers either do not dissolve or do not fullydissolve. To overcome this problem, the inventors discovered that byproviding a light oil, such as a silicone oil (e.g., silicon oil), withreconstitution reagents, they were able to direct the reconstitutionreagents toward the centers of chambers, thus improving reconstitutionof dried process materials. The oil was also found to have a “squeegee”effect, in which the oil essentially sweeps along the walls of achamber, thereby causing all or substantially all of a substance to bemoved into an adjacent chamber. This is particularly critical in unitdose applications that are sensitive to changes in the amounts orconcentrations of process materials. Oil was also found to contribute tobetter mixing of substances by concentrating aqueous substances near thecenters of chambers, which, in combination with the squeegee effect,ensures that more of the substances being mixed are transferred betweenchambers. An additional benefit of oil is its coating ability, whichprevents or interferes with substances sticking to the surfaces ofchambers. As an alternative to oil, other inert, immiscible liquidshaving similar advantages may be used.

It should be mentioned here that an advantage of the receptacle 10embodying aspects of the present invention is the ability, due to thenon-linear arrangement of the chambers, to reconstitute processmaterials in a non-sequential manner. That is, it is not necessary forthe first process material to be fully reconstituted beforereconstituting the second process material. The first process materialcan be reconstituted in chamber C22 and the second process material canbe reconstituted in chamber C28 at any time that is required orconvenient for performing a process, including simultaneously.

A pressure application mechanism physically presses upon chamber C30 toproduce fluid pressure that opens the seal closing portal 66 betweenchamber C30 and chamber C28, allowing fluid flow between the chambersand transferring the reconstitution reagent from chamber C30 to chamberC28 to dissolve the second process material contained therein. If theprocess material contained in chamber C28 is already in a liquidform—for example, if the enzyme reagent and detection probe are preparedin a liquid form or are reconstituted prior to loading them into thereceptacle—chamber C30 may be empty. For certain liquid processmaterials, it may be desirable to include a detergent, such as TRITON®X-100 (octylphenolpoly(ethyleneglycolether)_(n)), to prevent componentsof the process materials from sticking to the walls of the chamber.Furthermore, if the process materials provided in a liquid orreconstituted form are sensitive to electromagnetic radiation, then thechamber holding these process materials (e.g., chamber C28) may beconstructed using light-shielding materials.

It is often desirable and necessary to effect a mixing betweensubstances moved from one chamber into an adjacent chamber. For example,when moving a reconstitution reagent from chamber C30 into chamber C28to reconstitute a dried process material contained in chamber C28, it isdesirable to mix the reconstitution reagent and dried process material.In the illustrated embodiment, chamber C30 and chamber C28 arepositioned and oriented with respect to each other to facilitate gravityassisted mixing of the combined contents of the two chambers. Withgravity assisted mixing, a pressure mechanism is used to force asubstance (e.g., reconstitution reagent) from a lower chamber (e.g.,chamber C30) to an upper chamber (e.g., chamber C28). Gravity assistedmixing generally depends on at least one of the following mechanisms:(i) turbulence generated when a substance is forced through a relativelynarrow passage connecting adjacently positioned upper and lower chambers(or upper and lower regions of a chamber connected by a restrictedsection), where the substances in both the upper and lower chambers (orsections of a chamber) contain liquids; (ii) movement of the combinedliquids about the periphery of the upper chamber; and (iii)gravitational movement of the substance through the passage connectingthe upper and lower chambers. One advantage of gravity assisted mixingis that a pressure mechanism does not have to be associated with theupper chamber (or upper region of a chamber).

Chamber C34 contains a wash reagent which is used to remove unwantedmaterials from the sample processing procedure performed in chamber C26.The volume of wash reagent contained in chamber C34 in preferredembodiments is from about 400 μL to about 5,000 μL and most preferablyis from about 700 μL to about 2,000 μL. A pressure application mechanismpresses selected portions of chamber C34, thereby producing a fluidpressure that opens the seal closing portal 72, and forces wash reagentinto chamber C26, the sample processing chamber. As described above,chamber C34 includes an upper portion 38, a lower neck 40, a verticalsection 42, and a lateral section 44 extending toward chamber C26. Dueto the arrangement of chamber C34, it is believed that gravitationalforces assist in moving the wash reagent from the upper portion 38through the lower neck 40. In some embodiments, the instrument mayinclude passive means, such as a sponge or other compressible bodypositioned adjacent upper portion 38, to apply a continuous andrelatively mild pressure to the upper portion to further assist inforcing substance toward the lower neck 40. From the lower neck,pressure mechanisms, examples of which are described below, are used toforce the wash reagent—usually a portion at a time—through the verticaland lateral sections 42, 44 and then through portal 72 into chamber C26.Another pressure mechanism, an example of which is described below, ispositioned at lower neck 40 to function as a clamp for selectivelystopping further movement of wash reagent.

Chamber C36, when used for waste collection, is empty prior toperforming a procedure and is designed to contain the total wastematerial volume required by a procedure, including, for example, wastematerials containing sample material, wash reagent, rinse reagent, andother expended process materials (e.g., reagents). In general, thepreferred capacity for chamber C36 is about 2 mL when used as a wastechamber.

As explained above, portal 70 connects chamber C26 and chamber C36 andhas an upper orientation relative to chamber C26. This orientation wasdiscovered to be advantageous because bubbles which may be formed inchamber C26 during a separation procedure, possibly due to the presenceof detergent-based solutions, will naturally tend to rise and accumulateadjacent to portal 70 near the top of chamber C26. Thus,bubble-containing waste materials are more easily and efficientlytransferred to chamber C36 and, therefore, less likely to interfere withsubsequent signal detection steps. The location of portal 70 adjacentthe top of chamber C26 also helps to retain solid support particles inchamber C26 when waste material is removed during a sample processingprocedure, especially one involving the use of magnetically-responsiveparticles. As discussed more fully below, during a preferred sampleprocessing procedure, magnetically-responsive particles used to bindanalyte are immobilized when a magnetic field is applied to the contentsof chamber C26. Bubbles that form in chamber C26 during sampleprocessing will tend to collect near the top of chamber C26 and willgenerally not come into contact with the more centrally locatedmagnetically-responsive particles when the waste material is moved fromchamber C26 to chamber C36. If the connection between a sampleprocessing chamber and a waste chamber is other than at the top ofsample processing chamber, then at least some bubbles will remain in thesample processing chamber when the waste materials are moved from thesample processing chamber to the waste chamber. Additionally, at leastsome of the bubbles that form will likely pass over the immobilizedparticles and could impart a force strong enough to dislodge someparticles, thereby causing some of the particles to be transferred tothe waste chamber with the waste materials. The sensitivity andrepeatability of processes are thereby improved by the design of thechambers in the exemplified receptacle 10 because solid supportparticles are more likely to be retained in the designated sampleprocessing chamber during a separation procedure.

In the illustrated embodiment employing receptacle 10, chamber C26 (thesample processing chamber) is connected to six chambers, includingchamber C16 (the sample chamber), chamber C22 (a first process materialchamber), chamber C24 (the rinse reagent chamber), chamber C28 (a secondprocess material chamber), chamber C34 (the wash reagent chamber), andchamber C36 (the waste chamber). Prior to performing an assay or otherprocess in the receptacle, chamber C26 can be empty.

When receptacle 10 is placed in an automated instrument (describedbelow), chamber C26 is oriented so that a removable magnetic field canbe applied to the area of chamber C26. In one embodiment, the magneticfield is applied by an actuator which moves a permanent magnet to aposition adjacent to the chamber C26. Suitable magnets are those havinga holding force of about 4.0 lbs. each, such as those available fromBunting Magnets Co. of Newton, Kans. as Catalog No. N50P250250. In apreferred aspect of this embodiment, the magnet is moved into positionby a magnet actuation mechanism (described in more detail below) alongthe plane of the receptacle when the receptacle is placed into theautomated instrument. The magnet may be moved from any directionrelative to the receptacle. The magnet applies a magnetic field tochamber C26 and its contents of sufficient strength to retain magneticparticles in chamber C26 while the field is being applied. As will beappreciated by the person of ordinary skill in the art, when a permanentmagnet is used, the magnet must be movable to a location sufficientlydistant from chamber C26 to remove the effect of the magnetic field fromthe sample processing chamber, when desired. Thus, the magnet is locatedon a movable magnet actuation means which can be moved to at least twopositions: (1) an “on” position in which the magnet is adjacent tochamber C26 and sufficiently close to apply a particle-retainingmagnetic field to the chamber and its contents, and (2) an “off”position in which the magnet is positioned sufficiently distant fromchamber C26 such that no magnetic field of substantial strength isapplied to the chamber or its contents and any magnetic particlespresent in the chamber are not appreciably affected thereby.

Alternative means for applying the magnetic field to the desiredlocation may include selective activation of an electromagnet, which iseither located adjacent to chamber C26 during an assay or process by anautomated instrument or which is moved into such position prior toactivation. Still further means for selectively applying a magneticfield include a permanent magnet mounted on a platen that is movabletransversely with respect to the plane of the receptacle 10 into and outof magnetically affecting proximity to chamber C26. Any suitableactuating mechanism can be used to move the platen, such as a threadedrod operatively coupled to a suitable motor, an electronic linearactuator, or a solenoid. Such magnetic separation means are known per sein the art and can easily be modified by the skilled artisan to anyconformation or orientation of receptacle chambers.

As mentioned above, opening the seals blocking passages between chambersand then transferring substances between adjacently connected chamberscan be effected by pressure application mechanisms. The pressureapplication mechanisms of the automated instrument deliver a physicalforce to the outside of the receptacle at select locations and, inparticular, to the outside of individual chambers of the receptacle atpredefined instances as governed by a computer controller. In thecontext of the present invention, the term pressure applicationmechanism refers to any means for delivering a physical pressing forceto the external surface(s) of the receptacle. Preferably each pressureapplication mechanism comprises a compression pad with areceptacle-contacting surface. The compression pad is coupled to anactuator that moves the pad relative to the receptacle, generallyperpendicularly with respect to the face of the receptacle, selectivelyinto and out of pressing contact with the receptacle. Alternatively,roller bars or wheels may provide the physical force. The pressureapplication mechanisms may also have an additional function, such as toprovide a thermal change to the area adjacent to the actuator. When anactuator comprises a compression pad, the pad can be made of anymaterial suitable for exerting an appropriate force to the surface ofthe receptacle without damaging the receptacle. Typically, thecompression pad applies a pressure to either side of the receptacle,while the opposite side of the receptacle, when in the automatedinstrument, is supported against a wall. Thus, application of externalforce by the compression pad of the pressure application mechanismpinches the receptacle at a selected location to compress the receptacleat that location and force fluid movement in a chamber and/or effect atemporary separation of one chamber from another (or one portion of asingle chamber from another portion) by bringing the two sides of thereceptacle into fluid sealing contact with each other. Alternatively, apair of compression pads placed on opposing sides of the receptacle andboth moveable toward and away from the receptacle can pinch thereceptacle and its contents between them.

The functional architecture 700 of a system embodying aspects of thepresent invention is shown schematically in FIG. 2. The system operateson a reaction receptacle, or container, 10 (300, described below, SeeFIGS. 12A and 12B) which is schematically represented in FIG. 2 as aseries of interconnected rectangles (i.e., chambers) as if the containerwere shown in a transverse cross-section. Operation of the system iscontrolled by a computer or other microprocessor, represented in FIG. 2as the control and processing computer 730, which is programmed tocontrol operation of the system and processing of data. The system shownis a schematic representation; overall system operation may becontrolled by more than one computer. The control and processingcomputer 730 may reside within an instrument for processing thereceptacle, or it may be a separate, stand-alone computer operablyconnected—e.g., by serial cable, by network connection, or wirelessly—tothe instrument.

A first element of the system 700 is the substance movement controlsystem 701. Substance movement control system 701 both causes andcontrols movements of substances from chamber to chamber within thesystem. More specifically, the substance movement control system 701 mayinclude substance moving members 710, which apply a substance movingforce to individual chambers or to the contents of the chamber, passageblocking members 708, which selectively block and unblock portals orpassageways between individual chambers, and chamber partitioningmembers 706 which selectively divide individual chambers into two ormore sub-chambers by, for example, pressing against a flexible chamberwith a narrow-edged partitioning member to collapse a narrow portion ofthe chamber thereby forming two sub-chambers on opposite sides of thenarrow collapsed portion. Substance moving member 710, passage blockingmember 708, and chamber partitioning member 706 are moved, or actuated,by an actuator drive mechanism 704 which may comprise pneumatics,pneumatic pistons, hydraulics, motors, solenoids, etc. The actuatordrive mechanism 704 is controlled by an actuator controller 702,comprising a computer or other microprocessor device programmed tocontrol operation of the actuator drive mechanism 704 to regulatemovement, sequence, and timing of substance moving member 710, passageblocking member 708, and chamber partitioning member 706. The actuatorcontroller 702, in combination with the control and processing computer730, selectively activates the substance moving members 710, passageblocking member 708, and chamber partitioning members 706 in selectedsequences to control movement of fluids throughout the receptacle duringthe performance of an assay or other process performed in thereceptacle. In an alternate configuration, control of the actuator drivemechanism 704 may reside in the control and processing computer 730.

The architecture 700 may further include a temperature control system720, which may include heaters 724 and coolers 726 for selectivelyheating and/or cooling the contents of one or more chambers of thereceptacle that are in proximity to the heaters or coolers. It should beunderstood that the heaters 724 and coolers 726 may comprise a singlethermal element, such as a Peltier chip. Operation of the heaters 724and coolers 726 is controlled by a temperature controller 722, which maycomprise a computer or other microprocessor device programmed to controloperation (temperature, timing, and sequence) of the heaters 724 andcoolers 726, e.g., by regulating power to the heaters 724 and coolers726. Temperature sensors 728 detect the temperature of the heaters 724and coolers 726 and feed the temperature data to the temperaturecontroller 722 to control operation of the heaters 724 and coolers 726to achieve the desired temperatures. The temperature controller 722, incombination with the control and processing computer 730, controloperation of the heaters 724 and coolers 726 to provide the desiredtemperatures and sequences of temperature variations (e.g., thermalcycling) to perform an assay or other process within the receptacle. Inan alternate configuration, control of the heaters 724 and coolers 726may reside in the control and processing computer 730.

A detector system 712 is provided to detect an output signal from thecontents of one or more chambers, which signal may be indicative of thepresence and/or quantity of an analyte of interest. The detector system712 may comprise a fluorometric detector, or fluorometer, comprising anexcitation source 714 for generating an excitation signal. Theexcitation signal passes through optics and filters 718, and a resultingexcitation signal having a prescribed wavelength or other opticcharacteristic is directed at one or more of the chambers. Emissionsfrom the contents of the chamber pass through the optics and filters 718(which are not necessarily the same optic elements through which theexcitation signal passes) to a detector element 716, wherein the opticsand filters 718 may pass only an emission signal of a prescribedwavelength to be detected by the detector element 716. Control of theoperation of the detector system 712, as well as processing of the datacollected by the detector system, may be performed by the control andprocessing computer 730.

An automated instrument for performing a procedure on a sample incooperation with a multi-chambered receptacle, such as receptacle 10,and which embodies aspects of the present invention, is designated byreference number 100 in FIG. 3. The automated instrument 100 can be usedto perform all or a portion of the steps of a process in a single,multi-chambered receptacle, without the need for interaction by atechnician during the operation of the process or steps of the process.The instrument shown in FIG. 3 includes a processing unit 102 and a doorassembly 200. Certain components and surface panels are omitted from theprocessing unit 102, and a covering shroud is omitted from the doorassembly 200 in the illustrated embodiment so that the underlyingcomponents and features can be more readily observed.

Processing unit 102 includes a housing 104. It is noted that the toppanel of the housing is omitted in the figure. Housing 104 containselectronics, circuitry, and pneumatics for the operation of theinstrument 100. Barcode reader brackets 106 and 108 hold a barcodereader (not shown). In one embodiment, a barcode label is placed on thereceptacle 10, and the barcode reader situated on the instrument willread this barcode and provide information such as process instructions,expiration information, calibration information, and sampleidentification. Brackets 106 and 108 hold the barcode reader forhand-held reading of the receptacle barcode label.

A display panel 110 projects upwardly from the housing 104 and ispositioned and oriented to permit a user ready access to any controlswitches mounted on the panel and to readily view any displays mountedon the panel.

The housing 104 further includes a front portion 120 which carries manyof the functional components of the processing unit 102. The frontportion 120 of the housing 104 includes a pressure mechanism cluster 180(described in more detail below) mounted with an actuator plate 124,which may be formed (e.g., machined) from Delrin® or aluminum, which maybe coated with Teflon® (PTFE). A recess 130 formed in the actuator plate124 forms an opening for receiving and holding a receptacle 10 placed inthe instrument unit 100 prior to closing the door assembly 200. The doorassembly is hinged or otherwise mounted with respect to the frontportion 120 of the housing so as to permit movement of the door assembly200 between a receptacle-receiving, open position and a closed position.Latches or other similar mechanism (not shown) may be provided toreleasably hold the door assembly 200 in the closed position withrespect to the housing 104. More specifically, the latch or othermechanism may be provided to hold the door assembly 200 in the closedposition but be adapted to release the door and permit its movement tothe open position upon application of a moderate amount of door openingforce.

To begin a process, the receptacle 10 is placed in the instrument 100,and the door assembly 200 is then closed. The receptacle 10 may includeone or more registration features, such as alignment holes 74 and 75,which cooperate with mating features within the instrument, such ashooks or alignment pins (not shown) provided within the instrument 100for properly positioning and orienting the receptacle with thereceptacle-receiving opening of the instrument. As an alternative to theexemplary embodiment shown, an instrument incorporating aspects of thepresent invention may include a slot or other opening into which thereceptacle is operatively placed, and a pivoting door assembly may beomitted. Sample material is preferably transferred to the receptacleprior to its placement in the instrument. Adding sample material to thereceptacle before positioning the receptacle in the instrument minimizesopportunities for the instrument to be contaminated with spilled samplematerial.

Details of the door assembly 200 are shown in FIGS. 3 and 5. In thefigures, a shroud, or housing, preferably covering portions of the doorassembly is not shown so as to permit the underlying components of thedoor assembly to be seen.

FIG. 5 shows the front side of the door assembly 200, i.e., the side ofthe door assembly 200 that faces the processing unit 102 and thereceptacle when the door assembly is closed. The door assembly 200 mayinclude one or more thermal zones for heating and/or cooling regions ofthe receptacle that are in proximity to the thermal zones. The exemplarydoor assembly 200 shown in FIG. 5 includes five thermal zones 260, 262,264, 266, and 268. The thermal zones are specifically located to provideheating and/or cooling to one or more specific chambers of thereceptacle. In the illustrated embodiment, thermal zone 260 coverschamber C16 and neck portion 51. Thermal zone 262 covers chambers C18and C20. Thermal zone 264 covers chambers C34, C32, C30, and most ofC36. Thermal zone 266 covers chamber C28. Thermal zone 268 is located onthe magnet translation mechanism 208 (described in more detail below)and covers chamber C26 and parts of chambers C22 and C24.

One or more thermal zones may be used to provide localized heatingand/or cooling to one or more specific chambers of the receptacle or toprovide controlled and stable ambient temperatures within theinstrument. The ambient temperature may be any convenient temperaturefor optimal performance of a process or particular steps of a process,as described above. For example, the ambient temperature may be in therange of about 20° C. to about 40° or in the range of about 25° C. toabout 37° C.

The thermal zones are preferably designed to rapidly heat (and/oroptionally rapidly cool) an area of the receptacle and its contents toany desired temperature. Rapid temperature changes may be needed forprocesses requiring thermal cycling, such as PCR amplificationreactions. Ideally, the thermal zones will have a high temperature rangeto accommodate variations between processes to be performed. Therefore,the temperature range of the thermal zones is preferably from about 5°C. to about 95° C. for water-based fluids, and may be much greater fornon-aqueous fluids, such as those containing oil.

The portions of thermal zones 260, 262, 264, 266 and 268 visible in FIG.5 are conductor plates made from a thermally conductive material, suchas copper or aluminum, for conducting thermal energy (heating and/orcooling) from a thermal energy source, such as a Peltier thermoelectricdevice, to the receptacle 10. The exposed surface of each conductorplate, as shown in FIG. 5, has a size and shape conforming to the areaof the receptacle intended to be affected by the thermal zone. Eachconductor plate is mounted within a conforming opening formed in blocksof non-conductive material, which provide thermal separation between theconductor plates. Preferably, the exposed surfaces of each of theconductor plates for thermal zones 260, 262, 264, 266, and 268 and theexposed surfaces of the separating blocks are coplanar, together forminga flat surface that contacts the side of the receptacle 10 when the doorassembly 200 is in the closed position.

The conductor plate of each thermal zone is in thermal contact with asource of thermal energy for conducting heating or cooling energy fromthe source to the exposed surface of the plate and then to thereceptacle. In one embodiment, the source is a thermoelectric module,otherwise known as a Peltier device. In a preferred embodiment,thermoelectric units are mounted within the door assembly 200 in thermalcontact with the thermal zones. Suitable Peltier devices includeTEC1-12708T125 for thermal zone 264, TEC1-12705T125 for thermal zones260 and 262, and TES1-12704T125 for thermal zones 266 and 268, allavailable from Pacific Supercool Ltd., Bangkok, Thailand.

Thermal insulation, such as foam insulation, may be provided around thethermoelectric modules and between portions of the conductor plates. Asis generally known by persons of ordinary skill in the art, means may beprovided within the door assembly 200 for dissipating excess heat awayfrom the source of thermal energy, such as one or morethermally-conductive heat sinks which may be combined with one or morefan mechanisms for generating a convective airflow with respect to theheat sink(s).

The thermal zones 260, 262, 264, 266, and 268 are under microprocessorcontrol for controlling the magnitude and duration of the thermalconditions, including thermal cycling where indicated, affected by thethermal zones. And one or more of the thermal zones can be deactivatedduring a test in which heating and/or cooling in the area(s) of theinactive thermal zone(s) is not required. Accordingly, control of thethermal zones can be configured to accommodate a variety of differentprocess requirements.

To improve heat transfer to particular chambers, it was found that theuse of oil or other inert substance can reduce the volume of air (a verypoor thermal conductor) in a chamber and, simultaneously, increasechamber pressure. Increased chamber pressure can facilitate greatercontact between chambers and corresponding conductor plates, so that thecontents of the chambers are more completely and rapidly heated.

The magnet translation mechanism 208 is constructed and arranged to movea magnet—including a single permanent magnet, a cluster of permanentmagnets, and/or one or more electromagnets—relative to a chamber inwhich a magnetic separation procedure is being performed (e.g., chamberC26), referred to, for the purposes of this explanation, as the magneticseparation chamber. More specifically, the magnet translation assembly208 is constructed and arranged to move the magnet with respect to themagnetic separation chamber between: (1) an “on” position in which themagnet is sufficiently close to the magnetic separation chamber so thatthe magnetic field generated by the magnet will have a sufficient effecton the contents of the magnetic separation chamber to substantiallyimmobilize any magnetically-responsive materials within the magneticseparation chamber; and (2) an “off” position in which the magnet issufficiently removed from the magnetic separation chamber so that themagnetic field generated by the magnet will have an insufficient effecton the contents of the magnetic separation chamber to substantiallyimmobilize any magnetically-responsive materials within the magneticseparation chamber.

In the embodiment shown in FIG. 5, the magnet translation mechanism 208includes a magnet carrier which supports a magnet or cluster of magnetsand an actuator coupled to the carrier for moving the carrier up anddown relative to the door assembly 200 between the on and off positions.In the illustrated embodiment, the magnet translation mechanism 208caries a cluster of three magnets 210, with a magnet being omitted fromthe top or “12 o'clock” position on the mechanism 208. The 12 o'clockposition is closest to the portal 70 connecting the magnetic separationchamber 210 with the inlet 48 of the waste chamber C36. By omitting amagnet from this position, an accumulation of magnetic particles at thisposition is avoided. This helps minimize the number of magneticparticles inadvertently carried into the waste chamber C36 during therinse and wash steps of the magnetic separation procedure.

Referring to FIG. 4, the compression pads of pressure mechanism cluster180 are positioned in a pattern conforming to the location of thechambers and fluid pathways of the exemplary receptacle 10 shown in FIG.1 and are shaped to perform various of the process-related functionsdescribed herein. The automated instrument activates appropriatepressure mechanisms, magnets, and/or thermal zones in appropriatesequences, as controlled by an internal microprocessor controller.

The pressure mechanism cluster 180 is installed within the actuatorplate 124 and is shown schematically in FIG. 4. The cluster 180 includesa plurality of individual compression pads constructed and arranged forreciprocal movement transversely to the outer surface actuator plate 124to selectively apply pressure to selected portions of the receptacle 10.The pressure mechanism cluster 180 includes a plurality of compressionpads sized and arranged to align with various chambers and portals ofthe receptacle 10. Each compression pad includes a head operativelyattached to a reciprocating pneumatic actuator, a magnetic actuator,solenoid, or other suitable mechanical, electro-mechanical or otheractuator (not shown) for moving the pad out into compressing engagementwith a corresponding portion of the receptacle 10 and back into itsstowed position.

Compression pad P51-1 is positioned so as to align with a top portion ofthe neck 51 of the receptacle 10. Compression pad P51-2 is positioned soas to align with a lower portion of the neck 51 of the receptacle 10where the neck 51 enters chamber C16 Compression pads P16-1, P16-2,P16-3 and P16-4 are all positioned so as to align with differentportions of chamber C16 of the receptacle 10. Compression pad P16-1 isthe bottom compression pad for chamber C16, compression pad P16-2 is thetop compression pad for chamber C16, compression pad P16-3 is thedivider for chamber C16, and compression pad P16-4 is the frontcompression pad for chamber C16.

Having multiple pads P16-1, P16-2, P16-3 and P16-4 combined with a largechamber C16, which may be employed as the sample chamber, allowsflexibility in the size of the sample to be assayed. Divider pad P16-3can be used to partition the chamber C16 into two smaller chambers. Notethat chambers C26 and C28 are much smaller than chamber C16 and, thus,it is self-evident that the entire contents of chamber C16, if filledsubstantially to capacity, would not fit within chamber C26 and/orchamber C28. For some applications, a relatively large volume of samplematerial may be required to ensure that there is a detectable amount ofan analyte, if present in the sample, but subsequent chambers, such aschambers C26 and C28, for processing the sample cannot accommodate sucha large volume of sample material. The multiple pads adapted to compressdifferent portions of chamber C16 allows the sample to be moved fromchamber C16 to chamber C26 one portion, or aliquot, at a time.

Compression pads P18-1 and P18-2 are positioned so as to align with thechamber C18. Compression pad P18-1 is the rear compression pad forchamber C18 and compression pad P18-2 is the front compression pad forchamber C18.

Compression pad P20 is positioned so as to align with chamber C20.Compression pad P22 is positioned so as to align with chamber C22.Compression pad P24 is positioned so as to align with chamber C24.Compression pad P30 is positioned so as to align with chamber C30.Compression pad P32 is positioned so as to align with chamber C32.

Note that in the illustrated embodiment, there are no compression padsassociated with chamber C28 or with region 50 of chamber C36 or region38 of chamber C34. The instrument may, however, include other mechanismsfor imparting forces onto the chambers, or portions thereof, asdescribed in more detail below. Moreover, the illustrated embodiment isexemplary, and other embodiments encompassing aspects of the presentinvention may provide compression pads for chamber C28 as well asregions 50 and/or 38 or may omit compression pads for other chambersand/or regions thereof.

Compression pads P34-1, P34-2, and P34-3 align with the lateral section44 of chamber C34. Compression pad P34-1 is the #1 wash compression pad,compression pad P34-2 is the #2 wash compression pad, and compressionpad P34-3 is the #3 wash compression pad.

Compression pad P34-4 is the #4 wash compression pad and aligns with thevertical section 42 of the wash reagent chamber C34.

Compression pad P34-5 is the #5 wash compression pad and aligns with thelower neck 40 of the wash reagent chamber C34. The #5 wash pad headP34-5 further includes parallel raised ribs P34-5 a extending across thecompression pad. Ribs P34-5 a provide a tight compressive seal forclosing off the neck portion 40 of receptacle 10 to prevent fluid flowfrom the upper portion 38 of the wash reagent chamber 34 into thevertical section 42 and lateral section 44 of chamber 34. Compressionpads P36-1, P36-2 and P36-3 are aligned with the vertical inlet 48 ofchamber C36. Note that compression pad P36-3 is wider than thecompression pad P36-1 and P36-2 so that a portion of the compression padP36-3 covers the neck 46 of chamber C36. Compression pad P36-1 is the #1waste compression pad, compression pad P36-2 is the #2 waste compressionpad, and pad P36-3 is the #3 waste compression pad.

Compression pad P72 is a clamp aligned with portal 72. Similarly,compression pad P70 is a clamp that aligns with portal 70, compressionpad P62 is a clamp that aligns with portal 62, compression pad P58 is aclamp that aligns with portal 58, compression pad P60 is a clamp thataligns with portal 60, compression pad P66 is a clamp that aligns withportal 66, compression pad P68 is a clamp that aligns with portal 68,compression pad P56 is a clamp that aligns with portal 56, andcompression pad P54 is a clamp that aligns with portal 54. Compressionpad P64 is a clamp aligned with portal 64.

Compression pad heads may be formed from a black acetal resin sold underthe brand name Delrin® by DuPont of Wilmington, Del.

Compression pad P26 is positioned so as to align with chamber C26. In apreferred embodiment, pad P26 is coupled to a screw actuator or otherrelatively slow-moving actuator. A screw actuator provides slow andsteady compression, rather than the abruptly-applied compressive forcesgenerated by pneumatically actuated compression pads. This controlledmotion can provide several advantages. For example, a screw actuatorallows the user to control the rate and extent to which the compressionpad P26 moves, thereby making it possible to limit or prevent turbulencewithin a chamber being compressed. Avoiding turbulence is desirablewhen, for example, using detergent-based reagents that are prone tobubble under turbulent conditions or when removing wash reagent from achamber during a magnetic separation wash procedure. While the washreagent is being removed from a chamber containing immobilized,magnetically-responsive particles, turbulence within the chamber cancause the particles to become dislodged and, thus, to be washed awayinto a waste chamber. The controlled movement of the compression pad P26can also help to prevent over-compressing a chamber, which can result inpeeling apart or rupturing a wall of a chamber.

FIGS. 12A and 12B shows another receptacle 300 in accordance with thepresent invention. Like receptacle 10 described above, receptacle 300includes a generally planar vessel having flexible top and bottom sheetsformed from thin flexible materials, such as foils and/or plastics, anddefining a pouch-like vessel. The receptacle 300 has an upper edge 302and a lower edge 304 that indicate the preferred orientation of thereceptacle during use and define an upper direction and a lowerdirection. An exemplary receptacle of the type shown in FIG. 12A hasdimensions of about 5.5 inches by about 3.4-4.0 inches and is about 0.4inches thick (when filled with sample and process materials), but may beof any dimensions suitable for manual manipulation or for use with anautomated system, similar to the one described herein. Preferredmaterials for constructing receptacle 300 are the same as thosedescribed above for receptacle 10. Receptacle 300 includes an inlet port306 for loading a sample material, or other substance, into thereceptacle 300. Receptacle 300 includes nine chambers C320, C322, C324,C328, C332, C334, C336, C338, and C340.

As shown in FIG. 12A, the receptacle 300 may include a rigid frame 380comprising vertical portions 381 and 383, a top horizontal portion 384,and a bottom horizontal portion 385. A panel 382 may receive anidentifying label, such as a bar code or other human or machine readableindicia. The information carried on such label may include lot number,serial number, assay type, expiration date, etc.

A projecting tab 386 projects above the top portion 384 and provides anappendage for grasping the pouch 300 and inserting it into an instrumentand removing it from the instrument. A port cover 388 (e.g., a one-wayvalve) is provided for introducing sample into the sample chamber C328Athrough inlet channel C328C. Frame 380, including the projecting tab 386and sample cover 388, are preferably formed from a suitably rigidmaterial such as plastic.

Further details of the receptacle 300 are shown in FIG. 12B, which is anexploded view of the receptacle. Frame 380 comprises rear framecomponent 380 a and front frame component 380 b between which issandwiched a flexible pouch 301. Rear frame component 380 a includesvertical portions 381 a, 383 a, a top horizontal portion 384 a, a bottomhorizontal portion 385, and projecting tab 386 a. Front frame component380 b includes vertical portions 381 b, 383 b, top horizontal portion384 b, and projecting tab 386 b, but does not include a bottomhorizontal portion.

Each frame component 380 a and 380 b may be injection molded, and thetwo components may be connected to one another in a frame assembly byultrasonic welding. The flexible pouch portion 301 of the receptacle 300positioned and secured in the frame 380 by pins on the frame components380 a, 380 b extending through holes formed in the periphery of thepouch.

In an exemplary use of the receptacle 300, the chambers can be filledwith substances needed to perform a binding reaction. For example,sample material may be loaded into chamber C328 through inlet port 306.Chamber C328 consists of an upper region C328A and a lower region C328Bconnected by a restricted section 364 that can be closed by a pressureapplication mechanism so that the lower region is segregated from theupper region. Chamber C332 may be loaded with a sample processingreagent for binding and immobilizing an analyte present in the samplematerial on a solid support, the lower region C328B of chamber C328 may,in addition to receiving sample material, function as a sampleprocessing region of chamber C328 for separating the immobilized analytefrom other components of the sample material, chamber C334 may be loadedwith a dried, first process material, chamber C340 may be loaded with areagent for reconstituting the first process material, chamber C322 maybe loaded with a dried, second process material, chamber C324 may beloaded with a reagent for reconstituting the second process material,chamber C336 may be loaded with a wash reagent, chamber C338 may beloaded with a rinse reagent for removing inhibitory components of thewash reagent, and chamber C320 may function as a waste chamber forreceiving and storing waste substances in relative isolation from otheraspects of the reaction. In addition to containing the second processmaterial, a lower region C322B of chamber C322 may also function as adetection region of chamber C322 for detecting a signal or change in areaction mixture that is indicative of the presence of at least oneanalyte of interest in the sample material.

Chamber C320 is configured to receive waste materials from chamber C328and includes an initial, generally vertical inlet 370 extending fromchamber C328, an upper neck 372, and a collection region 374. Verticalinlet 370 is positioned generally above the lower region C328B ofchamber C328 and is connected to chamber C328 by means of portal 360positioned near the top of the lower region C328B of chamber C328. Thearrangement of chamber C320 relative to the chamber C328 allows forbubbles contained in chamber C328 to be transferred directly intochamber C320 when waste materials are moved from chamber C328 to chamberC320. Furthermore, because upper neck 372 is positioned above thecollection region 374 of chamber C320, waste material can be retainedwithin collection region 374 by force of gravity without the applicationof a clamp or other means for sealing the upper neck 372.

As illustrated in FIG. 12, in the interconnected chamber system of thereceptacle 300, chamber C324 is connected to the lower region C322B ofchamber C322 by portal 350, the lower region C322B of chamber C322 isconnected to chamber C328 by portal 356, chamber C340 is connected tochamber C334 by portal 344, chamber C334 is connected to the lowerregion C328B of chamber C328 by portal 346, chamber C332 is connected tothe lower region C328B of chamber C328 by portal 348, chamber C338 isconnected to the lower region C328B of chamber C328 by portal 362, andchamber C336 is connected to the lower region C328B of chamber C328 byportal 342. A wall 376 projects obliquely into chamber C336 forpreventing air bubbles that have collected in an upper portion ofchamber C336 from being moved through portal 342 and into chamber C328during a wash procedure. In one embodiment, each of the portals 342,344, 346, 348, 350, 356, 360, and 362 is temporarily closed by anopenable seal or other barrier to prevent fluid flow therethrough. Likereceptacle 10, receptacle 300 defines a non-linear arrangement ofchambers useful for performing complex procedures requiring orbenefiting from non-sequential processing of samples.

Chamber C322 includes the lower region C322B discussed above and anupper region C322A which are connected by a restricted section 358. Inthe illustrated arrangement, the combined substances of chambers C324and C322 (before or after being combined with substances from chamberC328) can be mixed in chamber C322 by moving the combined substancesback-and-forth between the upper and lower regions C322A, C322B ofchamber C322, while each of portals 350 and 356 is clamped by a pressureapplication mechanism to prevent substances from moving into chambersC324 and C328. Due to the relative orientations of the upper and lowerregions C322A and C322B of chamber C322, gravity assists in moving thecombined substances from the upper region C322A into the lower regionC322B as pressure being applied to the lower region C322B is removed.Thus, in the embodiment shown, there is no need for an external pressureto move substances from the upper region C322A to the lower region C322Bof chamber C322.

Receptacle 300 is processed in an instrument (not shown) having anarrangement of pressure application mechanisms—such as compressionpads—and thermal zones sized, shaped, and positioned to conform to thechambers of the receptacle 300 for selectively moving substances betweenchambers and for selectively providing heating and/or cooling to one ormore selected chambers.

A second embodiment of an instrument embodying aspects of the invention,and configured to process a receptacle 300, such as shown in FIGS. 12Aand 12B, is designated by reference number 1000 in FIG. 13. Instrument1000 includes a housing 1002 having a top portion 1002 and a bottom 1002b. Housing 1002 further includes a handle 1004. Handle 1004 includesopposed slots 1009 for holding a receptacle 300 during preparation.Instrument 1000 further includes an air intake 1008, preferably coveredby a suitable filter material, and an exhaust vent 1010. A status screen1012 displays status and other information useful to the operator, andoperation buttons may be provided, for example below screen 1012, asshown. A receptacle insert slot 1014 in the top portion 1002 a of thehousing is configured to receive a receptacle 300, as shown in FIG. 13.Receptacle insert slot 1014 is preferably convex so that spilled liquidwill run off the housing 1002, rather than into the slot. A slot coverslide 1016 can be manually manipulated after the receptacle 300 isinserted into the slot 1014 to provide a closure over the slot 1014 andmay again be opened to permit removal of the receptacle 300. Fan 1011provides cooling air for electronics and other components internal tothe housing 1002.

Looking into the interior of the housing 1002 in FIGS. 14 and 15, theinstrument 1000 includes an air compressor 1020 and an air reservoir1024. The instrument further includes a detector, such as fluorometer500 (described in more detail below), and a magnet actuator 1090 forselectively moving magnets into and out of operative position withrespect to the receptacle. Instrument 1000 further includes an airmanifold 1082 and an actuator plate 1080. Instrument 1000 may furtherinclude a coalescing air filter 1022. Aspects of the temperature controlsystem are also shown and include a thermal isolating frame 1048 and aheat dissipating system, including a fan 1070, a shroud 1068, and heatsink 1064. Fan 1070 draws air into the instrument through the air intake1008, and the heated air, after flowing over the heat sinks 1064, exitsthe housing 1002 through the exhaust vents 1010.

A pressure mechanism cluster of the instrument 1000 for applyingselective pressure to the receptacle 300 shown in FIG. 9 is shown inFIG. 16. The cluster is installed within the actuator plate 1080, whichmay be formed (e.g., machined) from Delrin® or aluminum, which may becoated with Teflon® (PTFE). As with cluster 180 described above, thecluster of FIG. 16 includes a plurality of individual compression padsconstructed and arranged for reciprocal movement transversely to theactuator plate 1080 to selectively apply pressure to selected portionsof the receptacle 300. The pressure mechanism cluster includes aplurality of compression pads sized and arranged to align with variouschambers and portals of the receptacle 300. Each compression padincludes a head operatively attached to a reciprocating pneumaticactuator, a magnetic actuator, solenoid, or other suitable mechanical,electro-mechanical or other actuator (not shown) for moving the pad outinto compressing engagement with a corresponding portion of thereceptacle 300 and then back into its stowed position.

In one embodiment, each compression pad is coupled to an air conduitformed in the manifold 1082, which directs pressurized air to the pad tomove the pad to an extended position. More specifically, eachcompression member is controlled by a solenoid valve which, whenengaged, connects pressurized system air to a pathway that goes to aportal where a pressure regulator (not shown) is installed, and theoutput of this regulator is connected back into another portal on themanifold 1082, which feeds the now-regulated & pressurized air to thecompression member. Pressure sensors may be provided for monitoringpressure within the system.

Compression pads P328-1 and P328-2 are aligned with lower and upperportions, respectively, of the sample chamber C328A of the receptacle300. Compression pad P328-3 is aligned with an upper neck C328C.Compression pad P364 is aligned with the restricted area P364 betweenchamber C328A and chamber C328B and provides a means for selectivelyopening or closing the restriction 364 for controlling fluid flowbetween chambers C328A and C328B.

Compression pad P338-2 is a circular pad aligned with an upper portionof the rinse chamber C338, and compression pad P338-1 aligns with alower portion of the chamber C338. Compression pad P362 aligns with theportal 362 connecting the rinse chamber with C338 with the magneticseparation chamber C328B and provides a means for selectively opening orclosing the portal 362 (after an initially-closed burstable seal hasbeen opened) for controlling fluid flow between the chambers.

Compression pads P320-1, P320-2, and P320-3 align with differentportions of the waste chamber C320. Compression pads P320-1 and P320-2align with lower and upper portions, respectively, of an inlet passageconnecting chamber C328B to the waste chamber C320 and are adapted formoving fluid up the passage into the upper neck 372 of the chamber C320.Compression pad P320-3 controls movement of fluid through the upper neck372 and, in particular, prevents fluid from flowing back from the wastechamber C320 toward the chamber C328B. Compression pad 360 is alignedwith the portal 360 connecting the waste chamber C320 with chamber C328Band provides a means for selectively opening or closing the portal 360to control fluid flow between the chambers.

Compression pad P356 is aligned with the portal 356 connecting chamberC322B and chamber C328B and provides a means for opening or closing theportal to control fluid flow between the chambers. Compression padP322-1 is aligned with chamber C322A, and compression pad P358 isaligned with the restricted section 358 connecting regions C322A andC322B of chamber C322. Compression pad P358 provides a means for openingor closing the restricted section 358 for controlling fluid flow betweenthe chambers.

Compression P322-2 is aligned with the chamber C322B. In one embodiment,lower region C322B of chamber C322 functions as a detection chamber fordetecting a signal or change in a reaction mixture that is indicative ofthe presence of at least one analyte of interest in the sample material.Accordingly, in some embodiments, compression pad P322 is configured toallow optical transmission through the compression pad, therebypermitting the detection of an optical signal emitted by the contents ofchamber region C322B.

Compression pads P324-1 and P324-2 are aligned with upper and lowerportions, respectively of the chamber C324. Compression pad P350 isaligned with the portal 350 connecting chamber C324 and C322B andprovides a means for opening or closing the portal to control fluid flowbetween the chambers.

Compression pads P332-1 and P332-2 are aligned with the upper end lowerportions, respectively of the chamber C332. Compression P348 is alignedwith the portal 348 connecting chamber C332 and chamber C328B andprovides a means for selectively opening and closing the portal 348 tocontrol fluid flow between the chambers.

Compression pad P340 is aligned with chamber C340, and compression padP334 is aligned with chamber C334. Compression pad P344 is aligned withportal 344 connecting chambers C340 and C334 and provides a means forselectively opening or closing the portal 344 for controlling fluid flowbetween the chambers. Compression P346 is aligned with portal 346connecting chamber C334 and C328B and provides a means for opening orclosing portal 346 for controlling fluid flow between the chambers.

Compression pad P336-1 is aligned with a portion of the wash chamberC336 and compression pad P336-2 is aligned with another portion of thewash chamber P336. Compression pad P336-2 aligns with a portion of thewash chamber P336 extending from an end of the oblique wall 376 and aperipheral side wall of the chamber C336 and provides a means fordividing chamber C336 into two sub-chambers. Compression pad P342 alignswith the portal 342 connecting chamber C336 and chamber C328B andprovides a means for selectively opening or closing the portal 342 tocontrol fluid flow between the chambers.

Compression pad P336-3 is aligned with a portion of wash chamber C336and is a bladder formed by a sheet of flexible, relatively non-porousmaterial, secured at its edges to a recess formed in the actuator plate1080. The bladder P336-3 is in communication with an air conduit formedin the manifold 1082 and may be controlled by a regulator. When filledwith air, the bladder P336-3 inflates (expands) to apply pressure to aportion of chamber C336 to displace wash fluid from the chamber. Thebladder P336-3 is preferred over a reciprocating compression pad forthis location because inflation of the bladder can be controlled toprovide a slow, steady displacement of chamber C336.

The function of the bladder P336-3 is to gently pressurize the washchamber C336 enough to move an aliquot of wash buffer reagent into thechannel area adjacent portal 342 The compress pad P336-2 has a singleraised thin-surface to hold the wash aliquot in place until it is movedto the chamber C328B. The bladder P336-3 is actuated much like any ofthe other compression members, controlled by a solenoid valve which,when engaged, connects the pressurized system air to a pathway that goesto a portal where a pressure regulator is installed, and the output ofthis regulator is connected back into another portal on the manifold1082 which feeds the now-regulated & pressurized air to the bladder. Asuitable operating pressure for the bladder P336-3 is approximately 10psi.

The pressure mechanism cluster may be covered by an elastomeric shield(See reference number 1081 in FIGS. 18 and 19) which stretches to permitthe compression pads to operate and which covers and protects thecompression pads, for example, from spilled fluids. The shield mayinclude one opening through which a detector (e.g., fluorometer 500) maydetect optical singles emitted from the contents of a chamber locatedadjacent the opening. The shield may be provided with non-stickproperties (e.g, a non-stick coating) to facilitate insertion andremoval of the receptacle 300 from the instrument 1000 after processing.

As shown in FIG. 17A, the pneumatic manifold 1082 is attached to theactuator plate 1080. The air reservoir 1024 is connected to the manifold1082 as is the magnet actuator 1090 and the detector 500. A window 1086permits viewing of a bar code or other label provided on the panel 382of the receptacle 300 (see FIG. 12A), and bar code reader 1088 isconstructed and arranged to read a bar code on the receptacle. Valves1084 (e.g., solenoid valves) control the pressure distribution to thevarious conduits of the manifold, each of which is connected to one ofthe pneumatic compression pads shown in FIG. 16.

More specifically, FIG. 17B shows a circuit diagram of the pneumaticsystem of the instrument 1000. The system shown in FIG. 17B differssomewhat from the structure shown in FIG. 17A. For example, the systemshown in FIG. 17B lacks an air reservoir. The pneumatic system includespump 1020 connected to a check valve 1030, a water trap 1032, and an airdryer 1034 (e.g., a desiccant device for removing moisture frompressurized air). A valve 1028 (e.g., a solenoid valve) is constructedand arranged for selectively disconnecting the pump from the pneumaticsystem by venting the pump to atmosphere “ATM”. A pressure sensor 1036detects the pressure in the system and may communicate with the controland processing computer 730 (See FIG. 2). The system may also include anaccumulator 1038. The system next includes the valves 1084 and thecompression members (e.g., as shown in FIG. 16). In FIG. 17B, only threevalves 1084 a, 1084 b, 1084 c and three associated compression membersP332-2, P332-1, and P348 are shown. As can be seen in the Figure, eachvalve 1084 a, 1084 b, 1084 c can selectively connect the associatedcompression pad to the pressure source (pump 1020 or accumulator 1038),connect the associated compression pad atmosphere “ATM” to vent thecompression pad (to remove pressure that might inhibit completeretraction of the compression pad), or block the compression memberbranch from the rest of the pneumatic circuit.

The magnet actuator 1090, shown in cross-section in FIG. 19, includes amotor 1092 which moves a magnet holder 1136 holding magnets 1132, 1134via a shaft 1094 coupled to a lead screw 1096. Magnet actuator 1090further includes cylinders 1110, 1112 which are coupled to an actuatorfitting 1140 for reciprocally moving a compression cup 1130. Motor 1092and cylinders 1110, 1112 are mounted atop a mounting block 1120 attachedto the manifold 1082.

More specifically, motor 1092 includes a shaft 1094 coupled to leadscrew 1096 by a coupler 1098 that is rotatably mounted within bearings1100, 1102. Lead screw 1096 is able to slide axially within coupler 1098and is threadably coupled to the magnet holder 1136 within which areheld a number of magnets, including magnets 1132, 1134 shown in thefigure. In a preferred embodiment, the magnet holder 1136 holds threesuch magnets. The magnet holder 1136 is disposed within a hollow portionof the compression cup 1130, which is disposed within a circular holeformed transversely through the actuator plate 1080. An end of the leadscrew 1096 inserted into the coupler 1098 has a square or other shapedcross-section that will prevent the lead screw 1096 from rotating withrespect to the coupler 1098. Furthermore, the magnet holder 1136 hasprojecting ridges or a non-circular peripheral shape that conforms tothe inner wall of the hollow compression cup 1130 to prevent the magnetholder 1136 from rotating within the compression cup 1130. Thus rotationof the lead screw 1096 by the motor 1092 causes correspondingtranslation of the magnet holder 1136.

In addition simply moving the magnet holder 1136 between on and offpositions, the motor 1092 can be controlled to vary the speed with whichthe magnet holder 1136 is moved from the on position to the off positionand vice versa as well as to vary positions between on and off. Thoseskilled in the art could imagine how, using a combination of speed andposition, the strength and rate of change of magnetic field strength canbe optimized to maximize magnetic particle retention.

Each of the cylinders 1110, 1112 includes a cylinder housing 1114 withinwhich is disposed a reciprocating piston 1116. (Note: Cylinders 1110 and1112 are identical; accordingly, only the features of cylinder 1110 arenumbered in the figure). A pneumatic port 1118 is provided for couplingthe cylinder 1110 to a source of air pressure. The pistons of each ofthe cylinders 1110, 1112 are coupled to the actuator fitting 1140.

The actuator fitting 1140 includes a circular center portion which fitsinto a portion of the same hole into which the compression cup 1130 fitsand two radial projections 1142, 1144 which fit into openings 1146,1148, respectively, formed into the actuator plate 1080 adjacent thecircular opening that receives the compression cup 1130. The pistons1116 are attached to the radial projections 1142, 1144.

As shown in the figure, the magnets 1132, 1134 carried in the magnetholder 1136 are in an “on” position. That is, the magnets are in closeproximity to the elastomeric shield 1081 covering the actuator plate1080, and thus are in close proximity to the chamber of the receptaclewithin which a magnetic separation procedure is being performed. Themagnets can be moved to an “off” position by rotating the lead screw1096 via the motor 1092 to translate the magnet holder 1136 away fromthe shield 1081 (i.e., to the right in the figure) within the hollowportion of the compression cup 1130. Reversing the rotation of the motor1092 and the lead screw 1096 extends the magnet holder 1136 back to the“on” position at the end of the compression cup 1130. Continued rotationof the lead screw 1096 will push the magnet holder 1136 and compressioncup 1130 out (to the left in the figure) against elastomeric shield 1081to apply a compressive force to a chamber adjacent the compression cup1130. Thus, the chamber is compressed to displace liquid from thechamber while the magnets are in the “on” position to hold and retainmagnetic particles within the chamber, for example during a rinse stepof the magnetic separation procedure.

When the compression cup 1130 is extended by the lead screw 1096 and themagnet holder 1136, the actuator fitting 1140, which is rigidly attachedto the compression cup (e.g., the two components are threaded together),also moves to an extended position (to the right in the figure). Thepistons 1116 of the cylinders 1110, 1112 move passively along with theactuator fitting 1140. When the magnet holder 1136 is retracted by thelead screw 1096, springs (not shown) within the cylinders 1110, 1112cause the actuator fitting 1140 and the compression cup 1130 to returnto the retracted position shown in the figure.

The magnet actuator 1090 also functions as a compression pad forapplying a compressive force to a chamber of the receptacle to force thefluid contents from the chamber when the magnets are in the “off”position. This is accomplished by turning the lead screw 1096 towithdraw the magnet holder 1136 to the “off” position (to the right inthe figure) and then pressurizing the pistons 1116 of the cylinders1110, 1112 to extend the pistons and thus extend the actuator fitting1140 and the compression cup 1130 (to the left in the figure) againstthe shield 1081, which will stretch and deflect in response to thereciprocating projection of the cup 1130, to compress a chamber adjacentthe compression cup 1130. As the actuator fitting 1140 is extended, themagnet holder 1136, which has been retracted back (to the right) intocontact with the actuator fitting 1140, will also be moved in thedirection of extension. To accommodate this movement of the magnetholder 1136, the end of the lead screw 1096 is able to slide with thecoupler 1098 (i.e., the lead screw 1096 “floats” within coupler 1098).Springs within the cylinder 1110, 1112 retract the actuator fitting 1140and compression cup 1130 when pressure is removed from the pistons 1116.

The temperature control system of instrument 1000 is shown in FIG. 20and includes thermal conductive elements 1040, 1042, 1044 disposedwithin a thermal isolating frame 1048. The thermal conductive elementsare preferably made from a thermally conductive material, such copper oraluminum, and the isolating frame 1048 is preferably formed from athermal insulating material, such as Ultem® or Delrin®. In theillustrated embodiment, each of the conductive elements 1040, 1042, 1044is sized and shaped so as to be in thermal communication with a regionof the receptacle 300 encompassing more than one chamber. Furthermore,at least a portion of each conductive elements 1040, 1042, 1044 is inclose proximity to at least a portion of an adjacent conductive elementsuch that a chamber encompassed by one conductive element is closelyadjacent to a chamber encompassed by the adjacent conductive element,and the two chambers are connected by a portal with no passagewayextending between the two chambers. Such close proximity betweenadjacent conductive elements without thermal crosstalk between theadjacent conductive elements is facilitated by the insulation providedby the isolating frame 1048.

The receptacle 300 is held in an operative position within theinstrument 1000 between the actuator plate 1080 and the isolating frame1048 (See FIGS. 14 and 15). The arrangement of the isolating frame 1048and the actuator plate 1080 within the instrument 1000 results in areceptacle-receiving gap therebetween, and that gap is dimensioned withrespect to the receptacle 300 such that when a chamber of the receptaclethat is adjacent one of the conductive elements 1040, 1042, 1044 isfilled with fluid, the chamber expands to increase the thermal contactbetween the surface of the chamber and the adjacent conductive element.

Peltier devices 1050, 1052, 1054 are positioned in thermal contact withconductive elements 1040, 1044, 1042, respectively. Temperature sensors1056, 1058, 1060 are positioned in thermal contact with the conductiveelements 1040, 1042, 1044, respectively, for sensing the temperature ofthe respective thermal conductive element. Sensors 1056, 1058, 1060 maycomprise RTD sensors, and are coupled to a controller (e.g., temperaturecontroller 722) for controlling operation of the Peltier devices.Heating elements other than Peltier devices, such as resistive foilheaters, can be used as well.

The Peltier devices 1050, 1052, 1054 (or other heating or coolingelements), are preferably mounted onto a heat sink 1062 which maycomprise an aluminum block having a first portion 1064 with a firstplanar side on which the Peltier devices are mounted and heatdissipating fins 1066 projecting from the opposite side. A shroud 1068partially covers the dissipating fins 1066 of the heat sink 1062, and acooling fan mounted within a fan housing 1070 is positioned for drawingair into shroud 1068 and past the heat dissipating fins 1066.

The Peltier devices 1050, 1052, 1054 can be selectively operated to heator cool the conductive elements 1040, 1044, 1042 and thereby heat orcool the contents of any chambers and portions of chambers of thereceptacle 300 that are in proximity to the respective conductiveelements. The conductive elements 1040, 1042, 1044, as well as theisolating frame 1048, are in a fixed position with respect to the pouch300. When the pouch 300 is inserted into the slot 1014 of the instrument1000, the pouch 300 is disposed in close proximity to the conductiveelements 1040, 1042, and 1044. When a chamber is filled with asubstance, the chamber of a flexible pouch will expand into theconductive element, thereby providing more complete physical, as well asthermal, contact with the conducting element positioned adjacent thatchamber.

The results of an analytical procedure performed with the receptacle 10or 300 and instrument 100 or 1000 are determined by measuring an opticaloutput of the sample, such as fluorescence or luminescence. Accordingly,an optical detector is provided with a lens projecting through opening176 formed in the front portion 120 of the processing unit 102. In theillustrated embodiment, the optical detector is a fluorometer 500.Alternative detectors could be readily adapted for use with theillustrated instrument, including detectors that sense electricalchanges or changes in physical characteristics, such as mass, color orturbidity.

Details of a fluorometer embodying aspects of the present invention areshown in FIGS. 6, 7, 8 a-c, and 9 a-c. The fluorometer 500 includes afront housing 502 and a rear housing 520 together mounted to a base 580.

Front housing 502 partially encloses an interior lens chamber 506 andincludes an upper barrel 504 having a generally cylindrical shape. Theupper barrel 504 extends into opening 176 formed in an actuator plate124 (see FIG. 3) within the instrument 100, or onto an opening formed inthe manifold 1082 of the instrument 3000 (see FIG. 14, 18). Fronthousing 502 further includes three mounting legs 510 for securing thefluorometer 500 to the actuator plate 124 within the instrument 100 orto the manifold 1082 of the instrument 1000.

The rear housing 520 is mounted, beneath the front housing 502, to thebase 580 by mechanical fasteners or the like. In the illustratedembodiment, the rear housing 520 includes four light conduits extendingfrom one end thereof to the opposite end thereof. In particular, therear housing includes a first emission conduit 522, a second emissionconduit 524, a first excitation conduit 526, and a second excitationconduit 528. Housing 520 is exemplary; the fluorometer 500 may includeone or more emission conduits and one or more excitation conduits.

Further details of the rear housing 520 are shown in FIGS. 6 and 7 a-c.Within housing 520, the two excitation conduits 526 and 528 areidentical and the two emission conduits 522 and 524 are identical.

Each excitation conduit 526, 528 includes a first portion 532 having across-section in the general shape of a right triangle with roundedcorners and a convexly rounded hypotenuse. The purpose of this shape isto limit the weight of the rear housing 520. The shape is merelypreferred; other cross-sectional shapes can be used for theconduits—including circular or rectangular—so long as the features ofthe conduit do not interfere with the passage of light. The excitationconduits 526, 528 further include a second portion 534 that is generallycylindrical in shape. A circular passage 536 connects the first portion532 with the second portion 534, and the diameter of passage 536 issmaller than that of second portion 534, thereby forming an annular lensshelf 540 within second portion 534. Finally, excitation conduits 526,528 include an O-ring seat 538 formed at the end of second portion 534.

Each emission conduit 522, 524 includes a first portion 552 having across-section in the general shape of a right triangle with roundedcorners and a convexly rounded hypotenuse. The emission conduits 522,524 further include a second portion 554 that is generally cylindricalin shape. A circular passage 556 connects the first portion 552 with thesecond portion 554, and the diameter of passage 556 is smaller than thatof second portion 554, thereby forming an annular lens shelf 560 withinsecond portion 554. Emission conduits 522, 524 also include an O-ringseat 558 formed at the end of second portion 554. Finally, a circularphotodiode seat 562 is superimposed within an end of the first portion552 of each of the emission conduits 522, 524.

Front housing 502 and rear housing 520 are preferably machined from 6061T6 aluminum and have a black anodized finish. Alternatively, the frontand rear housings could be molded or cast—either separately or as asingle, integrated unit, from any material that can withstand thetemperature environment within the instrument and will provideuninterrupted light conduits.

Base 580 includes a front printed circuit board (“PCB”) 582 and a rearprinter circuit board (“PCB”) 586. Front PCB 582 and rear PCB 586 areheld together in a fixed, spaced-apart relation by mechanical fasteners(e.g., bolts, screws) extending through cylindrical spacer elements 584.Details of exemplary circuits for the PCBs 582, 584 are described below.

With reference to FIG. 9 b, installed within the first excitationconduit 526 of the rear housing 520 are first excitation optic elements.The first excitation optic elements include a first light emitting diode(“LED”) 616 disposed at one end of the first portion 532 of the firstexcitation conduit 526 and mounted to the front PCB 582 of the base 580.A first excitation lens 618, which may be a collimating lens, is seatedon the lens seat 540 within the second portion 534 of the firstexcitation conduit 526. A first excitation filter 622 is positioned atthe end of the first excitation conduit 526 and is aligned in serieswith (i.e., along the optic axis of) the first excitation lens 618. Thefirst excitation filter 622 and the first excitation lens 618 areseparated from one another by a spacer 620, preferably made fromaluminum with a black anodized finish. The first excitation conduit 526,along with the associated optics elements, are referred to collectivelyas the first excitation channel.

Similarly, second excitation optic elements are installed within thesecond excitation conduit 528 of the rear housing 520. The secondexcitation optic elements include a second LED 632 disposed at one endof the first portion 532 of the second excitation conduit 528 andmounted to the front PCB 582 of the base 580. A second excitation lens634, which may be a collimating lens, is seated on the lens seat 540 ofthe second portion 534 of the second excitation conduit 528. A secondexcitation filter 638 is positioned at the end of the second excitationconduit 528 and is aligned in series with (i.e. along the optic axis of)the second excitation lens 634. The second excitation filter 638 and thesecond excitation lens 634 are separated from one another by a spacer636, preferably made from aluminum with a black anodized finish. Thesecond excitation conduit 528, along with the associated opticselements, are referred to collectively as the second excitation channel.

With reference to FIG. 9 c, first emission optic elements are installedwithin the first emission conduit 522 of the rear housing 520. The firstemission optic elements include a first photodiode 660 mounted within aphotodiode mount 648 disposed in the photodiode seat 562 within thefirst portion 552 of the first emission conduit 522 and mounted to thefront PCB 582 of the base 580. A first emission lens 650, which may be acollimating lens, is seated on the lens seat 560 of the second portion554 of the first emission conduit 522. A first emission filter 654 ispositioned near the end of the first emission conduit 522 and is alignedin series with (i.e. along the optic axis of) the first emission lens650. The first emission filter 654 and the first emission lens 650 areseparated from one another by a spacer 652, preferably made fromaluminum with a black anodized finish. Furthermore, the first emissionfilter 654 is separated from the end of the first emission conduit 522by an additional spacer 656, also preferably made from aluminum with ablack anodized finish. The first emission conduit 522, along with theassociated optics elements, are referred to collectively as the firstemission channel.

Similarly, second emission optic elements are installed within thesecond emission conduit 524 of the rear housing 520. The second emissionoptic elements include a second photodiode 662 mounted within aphotodiode mount 668 disposed in the photodiode seat 562 within thefirst portion 552 of the second emission conduit 524 and mounted to thefront PCB 582 of the base 580. A second emission lens 670, which may bea collimating lens, is seated on the lens seat 560 of the second portion554 of the second emission conduit 524. A second emission filter 674 ispositioned near the end of the first emission conduit 524 and is alignedin series with (i.e. along the optic axis of) the second emission lens670. The second emission filter 674 and the second emission lens 670 areseparated from one another by a spacer 672, preferably made fromaluminum with a black anodized finish. Furthermore, the second emissionfilter 674 is separated from the end of the second emission conduit 524by an additional spacer 676, also preferably made from aluminum with ablack anodized finish. The second emission conduit 524, along with theassociated optics elements, are referred to collectively as the secondemission channel.

A front housing cover disc 600 is disposed between the front housing 502and the rear housing 520 (see FIG. 6). The front housing cover disc 600includes a raised circular ridge 602 projecting slightly within the lenschamber 506 of the upper housing 502 (see FIGS. 8 b, 8 c). The fronthousing cover disc 600 further includes four circular light openings606, each being aligned with one of the light conduits 522, 524, 526,and 528 formed in the rear housing 520 when the cover disc 600 isinstalled.

A common lens 680 is housed within the lens chamber 506 of the fronthousing 502. In one embodiment, the fluorometer 500 includes only asingle, undivided common lens 680 which comprises the only optic elementof the fluorometer outside the excitation and emission conduits. In thiscontext, “undivided” means the common lens is made exclusively fromoptically transmissive material (e.g., glass) and includes no structurefor redirecting or impeding light, such as optically opaque structureembedded in and/or applied to the surface of the lens to physically andoptically divide the lens into two or more sub-parts.

An O-ring 682 is seated at an end of the lens chamber 506, and theraised circular ridge 602 of the front housing cover disc 600 projectsinto the lens chamber 506, pressing against the O-ring 682 to ensure alight-tight connection between the front housing 502 and the fronthousing cover disc 600. An O-ring 624 is provided within the O-ring seat538 at an end of the first excitation conduit 526 and compresses againsta rear side of the front housing cover disc 600 to provide a light-tightconnection. Similarly, an O-ring 640 is provided in the O-ring seat 538of the second excitation conduit, an O-ring 658 is provided in theO-ring seat 558 of the first emission conduit 522, and an O-ring 678 isprovided in the O-ring seat 558 of the second emission conduit 524.O-rings 624, 640, 658, 678 prevent light infiltration into the lightconduits 526, 528, 522, 524, respectively. The O-rings prevent lightinfiltration by compensating for the dimensional variations of themachined parts within the specified tolerance and also by compensatingfor the deformations induced by thermal factors.

In operation, excitation light signals are emitted by the light-emittingdiodes 616 and 632. The signals are preferably of a prescribedwavelength corresponding to a dye to be detected. Light from the diode616 is transmitted through the first excitation conduit 526 and impingesupon lens 618 which focuses at least a portion of the light to the firstexcitation filter 622. The first excitation filter 622 passes light ofonly a prescribed wavelength (or a prescribed range of wavelengths) andremoves undesirable wavelengths from the transmitted light. The filteredlight progresses through the common lens 680, which focuses at least aportion of the light out through the upper barrel 504 of the fronthousing 502, where the excitation light impinges upon a chamber (forexample, chamber C28) of a receptacle 10 within the instrument 100.Assuming the presence of a first analyte or group of analytes withinthat chamber, the dye of a first binding agent or group of bindingagents mixed with the sample and adapted to detect the presence of thefirst analyte(s) will fluoresce. A portion of the fluorescent emissionenters the upper barrel 504 and then travels through the common lens 680which directs at least a portion of the fluorescent emission into firstemission conduit 522. Light entering first emission conduit 522 passesthrough the first emission filter 654, which will filter undesiredwavelengths of emission light. The filtered emission light then travelsthrough the lens 650 and finally onto the photodiode 660, which willdetect the presence of light at the prescribed wavelength.

Similarly, excitation light signals emitted by diode 632 are transmittedthrough the second excitation conduit 528 and impinges upon lens 634which focuses at least a portion of the light to the second excitationfilter 638. The second excitation filter 638 passes light of only aprescribed wavelength (or a prescribed range of wavelengths) and removesundesirable wavelengths from the transmitted light. The filtered lightprogresses through the common lens 680, which focuses at least a portionof the light out through the upper barrel 504 of the front housing 502,where the excitation light impinges upon a chamber (for example, chamberC28) of a receptacle 10 within the instrument 100. Assuming the presenceof a second analyte or group of analytes within that chamber, the dye ofa second binding agent or group of binding agents mixed with the sampleand adapted to detect the presence of the analyte(s) will fluoresce. Aportion of the fluorescent emission enters the upper barrel 504 and thentravels through the common lens 680 which directs at least a portion ofthe fluorescent emission into second emission conduit 524. Lightentering second emission conduit 524 passes through the second emissionfilter 674, which will filter undesired wavelengths of emission light.The filtered emission light then travels through the lens 670 andfinally onto the photodiode 662, which will detect the presence of lightat the prescribed wavelength.

Light emissions detected by the photodiodes are converted to signalsthat can provide qualitative or quantitative information about thepresence or amount of an analyte or analytes in a sample using knownalgorithms. Examples of quantitation algorithms are identified in the“Uses” section hereinabove.

In the illustrated embodiment, the excitation conduits 526, 528 arelocated opposite each other, and the emission conduits 522, 524 arelocated opposite each other within the rear housing 520 to minimizebackground from excitation light passing through the emission filter.However, the excitation conduits 526, 528 and the emission conduits 522,524 could be located next to each other.

Fluorometer 500 includes two excitation channels and two emissionchannels, which permit the fluorometer to differentially detect two dyesor reporter moieties that are excited at different wavelengths. Lightemissions from these different dyes are generally quenched in theabsence of target (e.g., an analyte, a control, or amplification productthat is representative of the presence of either). Such dyes mayinclude, for example, N,N,N′N′-tetramethyl-6-carboxyrhodamine (“TAMRA”)and 6-carboxyfluorescein (“FAM”) or 6-carboxy-X-rhodamine (“ROX”) and2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (“JOE”). DABCYL isuseful quencher moiety for quenching light emissions from any of thesedyes in the absence of target. Thus, the instrument is capable ofdistinguishing between two different analytes or groups of analytes orof distinguishing an analyte or group of analytes from an internalcontrol. It is contemplated, however, that a fluorometer embodyingaspects of the present invention may include more or less than twoexcitation and emission channels, with the number of excitation channelsand the number of emission channels being equal.

The specific optical components selected for the excitation and emissionchannel(s) will depend on the wavelength of the dye fluorescence to bedetected. For the dye FAM, for example, a suitable LED for theexcitation channel is available from Kingbright Corporation of Brea,Calif. as Part No. L7113PBCH, and a suitable excitation filter for thesame dye is available from Semrock of Rochester, N.Y. as Part No.FF01-485/20-9.0-D. For the same dye, a suitable photo-detector for theemission channel is available from OSI Optoelectronics, Inc. ofHawthorne, Calif. as Model No. PIN-44DI, and a suitable emission filteris available from Semrock as Part No. FF01-531/22-9.0-D. For the dyeTAMRA, for example, a suitable LED for the excitation channel availablefrom Nichia America Corporation of Torrance, Calif. as Model No.NSPG500S, and a suitable excitation filter is available from Semrock asPart No. FF01-543/22-9.0-D. For the same dye, a suitable photo-detectorfor the emission channel is available from OSI Optoelectronics as ModelNo. PIN-44DI, and a suitable emission filter is available from Semrockas Part No. FF01-587/11-9.0-D.

Accordingly, a fluorometer embodying aspects of the present invention isable to excite and detect multiple, different signals (such as differentwavelengths) without moving with respect to the sample or without thedifferent excitation and emission channels moving with respect to eachother. Moreover, the arrangement of the fluorometer with respect to theactuator plate and the detection chamber of the receptacle carriedwithin the instrument, enables the fluorometer to direct excitationsignals at the detection chamber and detect emissions from the detectionchamber without the use of fiber optics. Moreover, the optic channels(excitation and emission) defined by the fluorometer are parallelthroughout there extents, and thus excitation light can be transmittedtoward the sample and emissions from the sample can be detected withoutthe use of reflective elements (e.g., mirrors) that redirectsubstantially all the light impinging on the element or lightcharacteristic separating elements that redirect a portion of a lightsignal having a first optical characteristic (e.g., wavelength) andtransmit another portion of the light signal having a second opticalcharacteristic, such as a dichroic beam splitter.

FIGS. 21-26 illustrate one embodiment of suitable circuitry. Thiscircuitry provides for local control of the fluorometer 500 withoperating modes selected, measurements made, and results reported inresponse to macro commands communicated remotely via a serial interface.

FIG. 21 represents circuitry comprising interconnection means and anumber of power supply circuits. FIG. 22 represents circuitry comprisingcontrol, processing and communication means, circuitry comprising meansto program and debug the processor, and a circuit that provides a stablevoltage reference. FIG. 23 represents circuitry comprising a voltagemeasurement circuit and a means to provide processor control of LEDintensity and modulation. FIG. 24 represents circuitry comprising theexcitation means (LEDs), an RF shield, and supplemental power filteringfor sensitive preamplifier circuits (described in the next figures).FIG. 25A and FIG. 25B represent two similar embodiments of a front-endamplifier circuit (the differences of which are explained later) whichconvert the modulated optical signal from the contents of a chamber intoa modulated electrical voltage. FIG. 26 represents a demodulationcircuit that converts the modulated and amplified signal into an analogvoltage proportional to the amplitude of the modulated optical signal.

This embodiment incorporates a modulation/demodulation scheme thatallows the circuit to reject the effects of varying background ambientlight whose wavelength falls within the band-pass range of the opticalfilters physically placed between the photodiodes D1 (660) (FIG. 25A)and D2 (662) (FIG. 25B) and the chamber being interrogated.Microprocessor U4 (FIG. 22) generates a clock (set at 275 Hz in thisembodiment) that is used to alternately modulate LED1 (616) and LED2(632) while controlling the polarity of the analog switch U7 (FIG. 26).By alternating the polarity of the analog switch U7 at the samefrequency and in phase with the modulation of the LED (either LED1 (616)or LED2 (632)), a matched transmitter/receiver pair is created. Onlythose optical signals arriving at the same frequency and in phase withthis clock will be amplified at full gain; all ambient light and otherlight signals modulated at a different frequency are suppressed.

In FIG. 21, J1 provides the main interconnection means to the circuit.Devices D1 and D6 protect the circuit by absorbing transient voltagesthat are applied to the circuit via this connection to externalcircuits. Integrated circuit U1 and associated components (C1, C3-C5,and R4-R7) form an adjustable voltage regulator that provides thepositive analog power supply for this circuit. Integrated circuit U16and associated components (C2, C51-C52, and R58-R59) form an adjustablevoltage regulator that provides the negative analog power supply forthis circuit. A separate +5V supply (U2 and associated components C7 andC10-C11) forms the digital power supply. Lastly, severalresistor-divider pairs are provided (R4/R5, R8/R9, R10/R11, and R56/R57)to translate the voltage level out of each supply to a voltage withinthe conversion range of the A/D converter found in the microprocessor(U4).

In FIG. 22, microprocessor U4 provides the primary control andprocessing means for the circuit. A number of capacitors (C15-C19)provide power supply bypassing for this device. Power-on reset of thecircuit is accomplished by circuitry incorporated within themicroprocessor, however, the microprocessor can be manually reset by useof the pushbutton switch SW1 (in combination with pull-up resistor R15).A diode D3 is provided to protect the circuit from potential staticdischarge associated with ungrounded contact with the reset switch.Crystal Y1 and associated components (C20 and C21) provide a stabletimebase and clock for the circuit. Communication between themicroprocessor (U4) and external circuits is accomplished by use ofintegrated circuit U5 and associated components (C23-C25), convertingTTL level serial signals in and out of the microprocessor to signals incompliance with the RS-232 standard. Programming and debugging of thecircuit is accomplished by use of the PROGRAMMING INTERFACE (componentsJ2, C22, and D4-D5). Visual indicators are provided to indicate “PowerOn” (components LED1 and R13) and “Status” (components LED2 and R14 withon/off control provided by the microprocessor U4). Lastly, a precisionvoltage reference circuit (components U3, C9, and C12-C13) is providedto establish a stable reference for the A/D converter (which isincorporated within the microprocessor U4) and to the external A/Dconverter U11 and D/A converter U12.

In FIG. 23, integrated circuits U11 and U12 provide an interface betweenthe analog circuits of the device and the microprocessor U4. Both ofthese devices are controlled by and communicate with the microprocessorU4 via its Serial Peripheral Interface (SPI). A/D CONVERTER U11 convertsthe differential analog signal out of the DEMODULATOR FILTER (FIG. 26)into a digital result with 24-bit resolution (signed, with approximately0.5 μA of resolution per bit). D/A CONVERTER U12 receives a digitalsetting from the microprocessor and sets a corresponding analog voltageon its output, a voltage which is then used to regulate LED current. TheDAC output voltage is connected to a resistor divider with a low-passfilter (components C45 and R37-R38) which lowers this output controlvoltage, resulting in the circuit being capable of controlling LEDcurrent over the range of 0-80 mA with 20 μA/bit resolution. Twoidentical circuits follow, one for FAM LED DRIVE and the other for TAMLED DRIVE.

In FIGS. 23 and 24, circuits are provided that directly regulate andmodulate the flow of current through the excitation LEDs (LED1 (616) andLED2 (632)). Power to the LEDs originates from the +12V supply. LEDcurrent flows first through resistor R3 that reduces the voltagepotential of the supply and, in conjunction with capacitor C3, providesfiltering of the switched current load to the modulated LED. Currentthen flows through the LED (LED1 (616) or LED2 (632)) and then throughthe drain source channel of a FET transistor (Q3 or Q4). Finally, theLED current passes through a feedback resistor (R53 or R54) thatgenerates a voltage proportional to the current through the LED.

Referring to FIG. 23, control of electrical current through the LED isachieved by use of a traditional feedback circuit comprised of anoperational amplifier (U13 or U14), FET transistor (Q3 or Q4), andfeedback resistor (R53 or R54). Control is achieved when the voltagepotential is equal at both inputs of the operational amplifier. Shouldthe voltage at the inverting input of the operational amplifier dropbelow the voltage at the non-inverting input, the voltage at the outputof the operational amplifier increases. This increase in output voltageis incident on the gate of the FET transistor, which then starts toconduct more electrical current. An increase in electrical currentthrough the feedback resistor results in an increase in voltage acrossthat resistor and a corresponding increase in voltage on the invertinginput of the operational amplifier, completing the feedback loop.

Additionally, to control switching (modulation or power on/off) of theLED, circuits are provided (Q1 or Q2 and associated components) thatforce the operational amplifier into and out of saturation, therebyswitching the controlling FET transistors (Q3 or Q4) off and on,respectively. In order to “inhibit” LED current, the voltage potentialat the gate of the FET transistor (Q1 or Q2) is taken several voltsbelow the voltage at the source terminal of the FET. This in turn allowscurrent to flow through the FET and into the circuit node formed at theinverting input of the operational amplifier, thereby causing thevoltage to rise to approximately 2.5V at that node. The output voltageof the operational amplifier subsequently drops to zero volts and theFET transistor (Q3 or Q4) is turned off, along with the respective LED.To “enable” LED current, the voltage potential at the gate of the FETtransistor (Q1 or Q2) is kept at a voltage equal to or slightly belowthe voltage at the source of the transistor. This keeps the transistorturned “off”, with no current flowing through the FET into the circuitnode at the inverting input of the amplifier. Voltage at the invertinginput of the operational amplifier is now equivalent to the voltageacross the feedback resistor (R53 or R54), and this voltage now tracksthe voltage applied by the DAC circuit to the non-inverting input of theoperational amplifier. LED current is controlled proportionally to thisapplied voltage.

In FIG. 23, LED health is monitored by the use of circuitry to probe thevoltage across and current passing through the LED. Resistors R51 andR52 provide a low impedance path between the A/D converter of themicroprocessor U4 (see FIG. 22) and the respective feedback resistors(R53 and R54); voltage measured by the A/D circuit across theseresistors is proportional to LED current. In addition, LED voltage canbe determined by use of the voltage buffering circuit formed byoperational amplifier U15 and associated components. A resistor dividercircuit (R47/R49 or R48/R50) follows the buffering amplifier to bringthe voltage down to a level within the conversion range of the A/Dconverter. LED voltage is calculated by taking the reduced voltage outof the buffer amplifier plus the voltage measured across the respectivefeedback resistor (which equates to the voltage across the droppingresistor R3) and subtracting these from the measured value of the +12Vsupply (with specific weighting for each measurement).

In FIG. 23, additional features are illustrated that improve circuitrejection of transient stimuli (both internally and externallygenerated). An RF shield E1 is provided to protect the circuitry foundin FIGS. 25A and 25B from electromagnetic and radio frequencyinterference. Two guard circuits (E8 and E9) are provided to preventelectrical leakage between the current carrying conductors to the LEDs(LED 1 (616) and LED2 (632)) and the sensitive circuits found in FIGS.25A and 25B. These guard circuits are comprised of exposed ground traceson the outside layers of the board, adjacent to and encircling exposedpads and traces connected to the LEDs. Lastly, four low-pass filters(R1/C1, R2/C2, R26/C20, and R27/C21) are utilized to provide additionalattenuation of power supply noise on the supplies used for thepreamplifiers (U1 and U2, FIGS. 25A and 25B).

Referring now to FIGS. 25A and 25B, a photodiode (D1 (660) or D2 (662))converts incident light (both background illumination and the modulatedfluorescent signal from the contents of the chamber being interrogated)into an electrical current which is supplied into the circuit nodeconnected to the inverting input of the operational amplifier (U1 orU2). Components D3 and R4-R6 (FIG. 25A; and D4 and R7-R9 in FIG. 25B)create a bias voltage on the anode of this photodiode; a higher biasvoltage increases dark current through the diode while decreasingphotodiode noise. Next, a compensation circuit is provided (U3A and U4Aand associated components) that generates an offsetting currentequivalent to the current out of the photodiode attributable to ambientlight. This compensation is vital as ambient light can create currentout of the photodiode that is many orders of magnitude greater than theelectrical current attributable to the modulated fluorescent signal.Without correction, any variation in the level of ambient light canresult in offsets to the measurement. In addition, without compensation,the current out of the photodiode associated with ambient light islikely to saturate the output of the preamplifier circuit. Lastly, atrans-impedance amplifier circuit is provided (U1 or U2 and associatedcomponents) that generates a voltage sufficient to source/sink anyadditional current out of the photodiode. The voltage out of thisamplifier (U1 or U2) is proportional to current out of the photodiodethat is attributable to the modulated optical signal from the contentsof the chamber being interrogated. Additionally, there is a small amountof signal associated with change in ambient background lighting (whichis filtered out by the subsequent demodulator circuit).

Due to the high gain of the trans-impedance preamplifier and the smallsignal being measured, the circuit described in the preceding paragraphcan be highly susceptible to drift as a result of changes in temperatureand humidity. To minimize these effects, a number of design and processprovisions are implemented in preparing circuit boards 582, 586. First,all circuit traces and components are located as far as possible fromother circuits. Second, the printed circuit solder mask has beeneliminated from underneath components R12-R15 and C16-C17. This providesa greater clearance between the circuit board and the components,enabling wash and rinse reagents to pass through during sampleprocessing procedures. Third, cylindrical resistors (MELF type) areselected for use at R12-R15 (again, maximizing clearance betweencomponents and the circuit board). Lastly, to minimize the amount ofcontaminants and residual flux remaining on the circuit board afterassembly, the board is first washed with saponifiers appropriate for thesolder/flux used in the soldering process, followed by a rinse withde-ionized water. Photodiodes D1 (660) and D2 (662) are preferablysoldered to the circuit board (after the above assembly process iscompleted) with a “no-wash flux” core solder. Residual flux remaining onthe circuit board after this last soldering process provides aprotective barrier and, therefore, is preferably not removed.

Referring again to FIGS. 25A and 25B, amplifiers U3B and U4B (andassociated components) provide additional amplification of the signal.In addition, feedback components R22 and C18 form a simple low-passfilter within that amplifier circuit (attenuating signals above 34 KHz).Concerning the compensation feedback circuit (identified in FIGS. 25Aand 25B as “SERVO FEEDBACK”), operational amplifiers U3A and U3B (andassociated components) are configured as integration amplifiers with acut-off frequency of approximately 5 Hz. The output voltage of theseamplifiers create a DC bias current that negates that portion of theelectrical current from the photodiode (D1 (660) or D2 (662)) that isattributed to background ambient light and other natural DC offsets inthe circuit. This results in an output signal (at U3B or U4B) that has azero DC voltage component, i.e., the signal is centered around 0V.

In FIG. 25A, components R28 and C22 (in combination with digital controlsignal “SERVO_ENABLE” from the microprocessor) are used to disable thecompensation amplifier (U3A only) from integrating at times when the TAMcircuit (FIG. 25B) is being utilized. This is because the output spectraof LED2 (632) (TAM) overlaps the band-pass of the optical filters infront of photodiode D1 (660). Without this disable feature, the FAMdetector/amplifier circuit (FIG. 25A) would integrate the excitationsignal used for the TAM circuit (FIG. 25B), resulting in longer circuitsettling times when the circuit is switched back to measure FAMresponse. Disabling of the integrating function in the FAM compensationcircuit (U3A and associated components) is accomplished by setting the“SERVO_ENABLE” output from the microprocessor U4 to active ground. Atother times, this digital signal from the microprocessor is tri-statedand components R28 and C22 have no effect on the operation of thecircuit.

Referring to FIG. 26, the entire circuit takes an incoming AC signal,modulated at the same frequency as the respective LED, and converts thissignal to a DC voltage (referenced to circuit ground) with amplitude sixtimes that of the peak-to-peak AC voltage. The two signals “PREAMP_A”and “PREAMP_B” (the outputs from the circuits in FIGS. 25A and 25B,respectively) are connected to the two contacts of a solid-state,single-pole, double-throw analog switch (U6). “FE_SEL” is the logicsignal that determines whether the signal “PREAMP_A” or “PREAMP_B” willbe connected through switch U6 to the subsequent analog switch U7.Analog switch U7 acts as a sort of buffer/inverter, alternatelyswitching its two inputs (the selected preamplifier output and circuitground) to either of the positive and negative inputs of the demodulatorfilter (operational amplifiers U8, U9, and associated components). Inthis implementation, the DEMODULATOR SWITCH (U7) is switched in phasewith the LED, such that when the LED is powered, the more positivesignal out of the preamplifier is switched to the positive input of thedemodulator filter (through R20) and the more negative signal isswitched to the negative input of the demodulator filter (through R21).Connections to the DEMODULATOR FILTER are reversed when the LED isturned off. In this manner, the maximum positive gain is attained fromthe demodulator and filter circuit.

Continuing with FIG. 26, the output of the analog switch U7 is connectedto a low-pass filter (comprised of components R20, R21, and C30) with acut-off frequency of approximately 9 Hz. Capacitor C30 of this filter ischarged to a DC voltage of amplitude approximately one-half that of thepeak-to-peak amplitude of the modulated signal coming out of thepreamplifier as a result of the switching of the preamplifier signalthrough analog switch U7. The subsequent active filter (operationalamplifiers U8A/B and U9A/B and associated components) comprises amulti-pole, low-pass filter with gain of approximately 12. In the firstpart of this filter, operational amplifier U8A/B (and associatedcomponents) form a differential amplifier with a DC gain of 4. Theoutput of this filter is passed through a low-pass filter (componentsR24, R25, and C35) with a cut-off frequency of approximately 3 Hz. Thesecond active stage of this filter is comprised of operational amplifierU9A/B (and associated components) which forms a differential amplifierwith a DC gain of 3. Components R32 and C36 form a feed-forwardcompensation path for the positive side of this filter while componentsR33 and C37 form a feed-forward compensation path for the negative sideof this filter. This filter is used to attenuate any and all signalsfrom the pre-amplifier that fall outside of a 10 Hz range around theoperating frequency of the LEDs (275 Hz in this example).

Finally, in FIG. 26, the outputs of the DEMODULATOR FILTER are fed intoa differential amplifier circuit (U10) with unity gain. Its function isto convert the voltage differential between the two signals out of thedifferential filter into a positive voltage referenced to circuitground. The two output signals “FLUORO+” and “FLUORO−” connect to theA/D converter discussed above.

In certain circumstances, it is necessary or desirable to move substanceout of a detection chamber of a receptacle. Under such circumstances, itmay be necessary to incorporate a chamber compression member with adetector, such as the fluorometer 500 described above. One suchembodiment of a compression member for incorporation with a detector isshown in FIGS. 10A and 10B. The compression mechanism, or detectoractuator, is designated by reference number 1150 and includes a bracket1152 connected to and projecting from the actuator plate 124. Acompression tube 1154 is positioned in front of the lens of the detector500 and extends through an opening in the actuator plate. Compressiontube 1154 may have a transparent window 1164 mounted at its distal endrelative to the detector 500. An actuating bar 1156 extends transverselyin opposite directions from the compression tube 1154. An actuatingmechanism, for example, a pneumatic piston represented by rod 1162connected to a pressure source by pneumatic line 1160, is carried on thebracket 1152 and engages the actuating bar 1156 to move the compressiontube 1154. A guide rod 1158 extends from the actuator plate 124 throughan opening in a bottom end of the actuating bar 1156.

Extending the actuating mechanism 1162 against the actuating bar 1156moves the compression tube 1154 to an extended position (to the left asshown in FIG. 10 b) to compress the chamber C against the door assembly200. The guide rod 1158 extending through the actuating bar 1156 helpskeep the compression tube 1154 in a straight orientation and helpsprevent skewing of the compression tube 1154 during movement of the tubeby the actuating mechanism 1162. Guide rod 1158 may be omitted ifskewing of the compression tube 1154 is not a concern.

An alternative mechanism for incorporating a compression member with adetector is shown in FIG. 11. In FIG. 11, detector 500 is mounted on atranslating mounting platform or sled 1172 having a base 1174 with guidepins 1176 and 1178 extending through longitudinal slots 1184 and 1182,respectively. A piston (for example, a pneumatic piston) 1180 causesreciprocal movement of the sled 1172 and the detector 500 and thusprovides a means for moving the entire detector into and out ofengagement with a receptacle chamber.

FIG. 18 shows a transverse cross section of an alternative embodiment ofa compression pad 1242 integrated with the signal detector 500. Thecompression pad 1242 comprises an actuator cup 1244 disposed within anopening 1083 formed through the actuator plate 1080. A transparentwindow, or detection lens, 1246 is positioned in front of the actuator1244 within an opening formed in the elastomeric shield 1081. Agenerally circular through bore 1186 is formed through the cup 1244 atan off center position with respect to an axis of symmetry of theactuator cup 1244, thus forming an upper portion 1188 of the actuatorcup 1244 that is thicker than a lower portion 1190 of the cup 1244. Areason for the off-center location is that there may be extra air in thedetection chamber during the detection process to help ensure betterthermal contact between the chamber and a heater element disposedadjacent the chamber and also helps with the fluidics transfer from thechamber of lower volumes of liquid. Because the extra air will cause theliquid to pool down towards the bottom of the “actuator area” (air risesto the top), the off-center detection lens and focal point of thefluorometer 500 are configured to read fluorescence in the lower portionof the detection area, where there will be more liquid (and thereforemore fluorescence). In an alternate implementation, for example, whenthe detection chamber if fully full of liquid, the bore 1186 may beformed through the center of the actuator cup 1244.

The actuator cup 1244 further includes a first radial lug 1192 and asecond radial lug 1194 extending from diametrically opposed positions onthe actuator cup 1244. Radial lug 1192 resides within a radial opening1196 extending from the opening 1083, and radial lug 1194 resides withina radial opening 1198 extending from the opening 1083. A circular blindhole 1200 extends from the radial opening 1196, and a circular blindhole 1202 extends from the radial opening 1198. Blind holes 1200 and1202 hold coil compression springs (not shown) which press against theradial lugs 1192 and 1194 to bias the cup 1244 in the retractedposition, as shown.

Detector 500 is mounted to the manifold 1082 over an opening 1085 formedthrough the manifold. First and second cylindrical projections 1204 and1206 extend from the manifold 1082 on opposite sides of the opening1085. An O ring 1208 is positioned on the cylindrical projection 1204,and an O ring 1210 is positioned on the cylindrical projection 1206.Cylindrical projection 1204 extends into a cup-like blind hole formed inthe radial lug 1192, and the cylindrical projection 1206 extends into acup-like blind hole formed in the radial lug 1194. An air pressureconduit 1212 extends into the cylindrical projection 1204 and exits atthe top of the projection. Similarly, an air conduit 1214 extends intothe cylindrical projection 1206 and exits from the top of theprojection. The actuator cup 1244 is moved from the retracted positionshown in FIG. 18 to an extended position (to the left as shown in thefigure), by applying pressure at the conduits 1212 and 1214, thuspushing the radial projections 1192 and 1194 up into the radial openings1196 and 1198, respectively, and moving the actuator 1244 to the left.The depth of the circular blind holes 1200 and 1202 accommodate thelength of the compressed springs (not shown) thereby permitting theradial lugs 1192, 1194 to move completely to the ends of the radialopenings 1196, 1198.

In the arrangements described heretofore, the thermal zone 266 (or 1044)is disposed on one side of the receptacle opposite the actuator plate1080 and the detector 500. Accordingly, with thermal energy beingapplied at only one side of the receptacle, a thermal gradient can becreated within the chamber (e.g. chamber C28) between the side of thechamber that is in close proximity or contact with the thermal zone 266and the opposite side of the chamber that is in contact with the lens1246 provided in the elastomeric shield 1081 (See FIG. 18). Certainreactions to be performed in chamber C28 have an optimal performancetemperature that is difficult to achieve when the receptacle is heatedfrom just one side and such a thermal gradient is created. TMAreactions, for example, have an optimal performance temperature of about42° C.±0.5° C. Therefore, the maximum temperature gradient across thecontents of the receptacle is preferably about 0.5° C. and morepreferably about 0.1° C., 0.2° C., or 0.3° C.

To minimize such potential thermal gradients, one aspect of theinvention is to apply thermal energy to a signal transmission elementdisposed between a receptacle and a detector so that thermal energy canbe applied to the receptacle by the transmission element itself. Thus,thermal energy is applied to both sides of the receptacle to therebyminimize any side-to-side thermal gradient within the receptacle. Oneembodiment of the invention includes a thermal element in thermalcommunication with a transmission element adapted to transmitelectromagnetic radiation from a sample contained within the receptacleto the detector, wherein the thermal element is constructed and arrangedto apply thermal energy to at least a portion of the transmissionelement that is in close proximity or contact with the receptaclewithout impeding the transmission element's ability to transmitsufficient signal to the detector.

FIGS. 32 and 33 show one embodiment of a transmission element 1244having incorporated therein a thermal element 1218. In the embodimentshown in FIG. 32, the transmission element disposed between a receptacle(not shown in FIG. 32) and a detector 500 is the actuator cup 1244 ofthe compression pad 1242, as shown in FIG. 18 and described above. Forpurposes of incorporating a thermal element with a transmission element,however, the transmission element need not be a movable compression pad,but may comprise a fixed, nonmoving element formed from a transmissivemedium and disposed between the detector 500 and the receptacle. Thetransmission element is constructed and arranged to transmitelectromagnetic radiation to be detected by the detector. For example,the transmission element may be an optic element adapted fortransmitting light signals, such as fluorescence or chemiluminescence.Such an optic transmission element can be made from any suitablematerial that will transmit light, such as transparent or translucentglass or plastic, including acrylic.

In the embodiment shown in FIG. 32, the thermal element comprises apass-through heater 1218 disposed within the bore 1186 of the actuatorcup 1244 against a lens 1216 disposed within an end of the bore 1186 soas to be generally flush with elastomeric shield 1081. Thisconfiguration is somewhat different from that shown in FIG. 18, in whichthe left-hand end of the actuator cup 1244 bears against and does notextend through the shield 1081 and which includes a lens 1246 disposedwithin the shield 1081. Lens 1216 may comprise an integral portion ofthe actuator cup 1244 or may comprise a separate element secured to orwithin the end of the bore 1186. The pass-through heater 1218 ispreferably a resistive heater incorporating one or more resistiveconductors embedded within or disposed upon a suitable film material,such as a polyester variant including Mylar® (biaxially-orientedpolyethylene theraphthalate (“boPET”)) or Kapton® polyimide. Thepass-through heater 1218 is preferably secured to the lens 1216 by asuitable, temperature-stable adhesive material.

In the illustrated embodiment of FIGS. 32 and 33, the pass-throughheater 1218 is in the form of an annulus with a central opening 1220through which electromagnetic radiation being transmitted through thetransmission element (e.g., actuator cap 1244) may pass. In oneembodiment, the pass-through heater 1218 has an inside diameter of 5 mmand an outside diameter of 12 mm. In an alternate embodiment, thepass-through heater 1218 need not be a closed annulus, but may be in theform of a partial annulus forming an arc adhered to a portion of thelens 1216 yet leaving a sufficient portion of the lens 1216 uncovered soas to permit electromagnetic radiation to be transmitted there through.

The pass-through heater 1218 is connected by suitable connectors, forexample wires (not shown), to a circuit having a power source and acontrol feature. The control feature may, for example, be part of theinstrument controller, such as the temperature controller 722, or thecontrol feature can be a resistive temperature detector (“RTD”) or athermistor.

When the pass-through heater 1218 is energized, its temperature changesand the change in temperature, e.g., heating, is transmitted through thelens 1216. Accordingly, a portion of the thermal energy generated by theheater 1218 will be applied to a receptacle that is in close proximityto or in contact with the lens 1216.

An embodiment of a power/control circuit to which the thermal elementmay be connected is designated generally by reference number 1230 inFIG. 38. In the embodiment shown, a thermal element 1238 is connected toa power supply 1232 and an RTD 1240 by means of a connector element1236. The circuit 1230 may also include a fuse 1234. The RTD functionsby comparing the current resistance of the RTD with a fixed resistancelevel. When the resistance level indicates that the temperature of thethermal element 1238 is not at the correct set point, a transistorcloses the circuit and turns the thermal element 1238 on. In oneembodiment of the invention, a 100-platinum RTD is used to provide thedesired accuracy level of 0.1 percent. Suitable RTD controllers areavailable from Minco of Minneapolis, Minn., part number CT325PD2C1.

The control circuit is preferably a closed-loop controller which mayemploy linear or pulse width modulation for controlling the temperatureof the thermal element. The thermal element may be set to a fixedtemperature when the instrument is powered up, or it can be adjustedduring operation of the instrument, thereby energizing it on only asnecessary to effect a desired temperature change. Adjusting thetemperature of the thermal element during operation has certainadvantages, such as limiting the impact on temperature-sensitivereagents that may be contained within the receptacle and conservingenergy, which becomes more critical when the instrument is batterypowered. Also, maintaining a constant temperature of the thermal elementmay lead to heat buildup within the instrument.

An alternate thermal element arrangement is shown in FIG. 34. In FIG.34, the thermal element is embodied in a heater film 1222 disposed alonga portion of the inner surface of a chamber defined by the bore 1186 ofthe actuator cup 1244 of the compression pad 1242. Again, thearrangement shown in FIG. 34 is exemplary. The heater film 1222 need notbe incorporated into a moveable compression pad 1242 but could bedisposed on the inner surface of a chamber defined within a fixed,non-moving transmission element. Suitable heater films include polyimidethermofoil flexible heaters or Kapton® polyimide film heaters. Heaterfilm 1222 is connected by suitable conductors (not shown) to apower/control circuit, such as circuit 1230 shown in FIG. 38. When theheater film 1222 is on, the temperature of the air, or other medium,within the chamber defined by the bore 1186 changes in response to theapplication of thermal energy by the energized film 1222, and a portionof that change in temperature is conducted through the lens 1216 to beapplied to a receptacle that is in close proximity to or in contact withthe lens 1216.

A further alternative embodiment of a thermal element incorporated intoa transmission element is shown in FIG. 35. In FIG. 35, a transmissivethermal element 1224 is secured within the bore 1186 of the actuator cup1244, preferably in contact with the lens 1216. Element 1224 maycomprise resistive elements disposed on or embedded within a film (e.g.a Mylar® screen) formed from a transmissive material. For example, theelement 1224 can be formed from a transparent or translucent material soas to transmit optical signals (e.g., chemiluminescence or fluorescence)therethrough. Exemplary, suitable transmissive heater elements includeThermal-Clear™ Transparent Heaters available from Minco. Transmissiveheater element 1224 is connected by suitable conductors (not shown) to apower/control circuit, such as circuit 1230 shown in FIG. 38. When theelement 1224 is switched on, its temperature changes, and a portion ofthat temperature change is conducted through the lens 1216 to be appliedto a receptacle that is in close proximity to or in contact with thelens 1216.

The arrangement shown in FIGS. 32, 33, and 35, in which the thermalelement is in direct contact with the lens 1216, are generally preferredover the arrangement shown in FIG. 34 where the thermal element isdisposed about the periphery of a chamber defined within thetransmission element because the temperature changes of the thermalelement are conducted through the lens 1216 more rapidly when thethermal element is in contact with the lens 1216, thus improvingefficiency and response time.

FIG. 36 is a partial transverse cross-section of a signal transmissionelement (movable actuator cup 1244 of compression pad 1242) attached tothe fluorometer 500 and incorporating a thermal element (pass-throughheater 1218) and a reaction receptacle 300 disposed so that chamberC328B is located between lens 1216 located at the end of actuator cup1244 and opposed thermal conductive element 1044 of the temperaturecontrol system. Compression pad 1242 is moveable in an axial directionbetween a retracted position and an extended position as described abovein the disclosure corresponding to FIG. 18. In FIG. 36, the actuator cup1244 of the compression pad 1242 is in a first, retracted position thatdoes not compress chamber C328B of receptacle 300. FIG. 37 is partialtransverse cross-section of the signal transmission element of FIG. 36,with the actuator cup 1244 of the compression pad 1242 shown in asecond, extended position compressing chamber C328B of the reactionreceptacle 300 against the thermal conductive element 300. In the secondposition, lens 1216 is in contact with chamber C328B, thereby providingthermal conduction between the actuator cup 1244, including heater 1218,and the chamber C328B.

EXAMPLES

Examples are provided below illustrating some of the uses of thereceptacles and systems provided herein. Skilled artisans willappreciate that these examples are not intended to limit the inventionto the particular uses described therein. Additionally, those skilled inthe art could readily adapt the receptacles and systems provided hereinfor use in performing other kinds of reactions, processes or tests.

The following examples provides a number of experiments that wereconducted to compare the sensitivity of a manual, real-timetranscription-mediated amplification (“TMA”) reaction with that ofautomated real-time TMA reactions using either liquid or driedamplification and enzyme reagents. TMA reactions are two enzyme,transcription-based amplification reactions that rely upon a reversetranscriptase to provide an RNase H activity for digesting the RNAtemplate after producing a complementary DNA extension product with anantisense primer or promoter-primer. Examples of TMA reactions aredisclosed in McDonough et al., U.S. Pat. No. 5,766,849; Kacian et al.,U.S. Pat. No. 5,824,518; and Becker et al., U.S. Pat. No. 7,374,885. Thetarget for this experiment was Chlamydia trachomatis 23S ribosomal RNA,referred to hereinafter as “the target nucleic acid.”

In each experiment, a “wobble” capture probe was used tonon-specifically bind the target nucleic acid in the test samples. Thewobble capture probe consisted of a 3′ region having a randomarrangement of 18 2′-methoxyguanine and 2′-methoxyuridine residues(poly(K)₁₈) joined to a 5′ tail having 30 deoxyadenine residues(poly(dA)₃₀). Complexes comprising the wobble capture probe and boundtarget nucleic acid were immobilized on magnetically-responsiveparticles having oligonucleotide tails consisting of 14 deoxythymineresidues (poly(dT)₁₄) derivatized thereon and then subjected to a washprocedure to remove interfering substances from the test samples.

After the wash procedure, the target nucleic acid was exposed to TMAreagents and conditions and the resulting amplification product wasdetected in real-time using a fluorescently labeled, molecular beaconprobe. See Kacian et al., U.S. Pat. No. 5,824,518; see also Tyagi etal., U.S. Pat. No. 5,925,517. The primers used for amplificationincluded an antisense promoter-primer having a 3′ target bindingsequence and a 5′ T7 promoter sequencer and a sense primer. Themolecular beacon probe was comprised of 2′-O-methyl ribonucleotides andhad an internal sequence for binding to the target nucleic acidsequence. The molecular beacon probe was synthesized to includeinteracting FAM and DABCYL reporter and quencher moieties usingfluorescein phosphoramidite (BioGenex, San Ramon, Calif.; Cat. No.BTX-3008) and 3′-DABCYL CPG (Prime Synthesis, Inc., Aston, Pa.; Cat. No.CPG 100 2N12DABXS). The probes and primers of this experiment weresynthesized using standard phosphoramidite chemistry, various methods ofwhich are well known in the art, using an Expedite™ 8909 DNA Synthesizer(PerSeptive Biosystems, Framingham, Mass.). See, e.g., Carruthers, etal., 154 Methods in Enzymology, 287 (1987).

Example 1 Manual Amplification Reactions

For this experiment, manual TMA reactions were set up in 12×75 mmpolypropylene reaction tubes (Gen-Probe Incorporated, San Diego, Calif.;Cat. No. 2440), and each reaction tube was provided with 125 μL of aTarget Capture Reagent containing 160 μg/mL 1 micron magnetic particlesSera-Mag™ MG-CM Carboxylate Modified (Seradyn, Inc.; Indianapolis, Ind.;Cat. No. 24152105-050450) derivatized with poly(dT)₁₄ and suspended in asolution containing 250 mM HEPES, 310 mM LiOH, 1.88 M LiCl, 100 mM EDTA,adjusted to pH 7.5, and 10 pmol/reaction of the wobble capture probe.Each reaction tube was then provided with 500 FL of a mixture containinga Sample Transport Medium (150 mM HEPES, 294 mM lithium lauryl sulfate(LLS) and 100 mM ammonium sulfate, adjusted to pH 7.5) and water in a1-to-1 ratio. The mixtures contained either 10⁵ copies of the targetnucleic acid (test samples) or no target nucleic acid (negative controlsamples). The reaction tubes were covered with a sealing card and theircontents mixed by vortexing for 15 seconds, and then incubated at 25° C.for 5 minutes in a water bath to facilitate binding of the wobblecapture probes to the target nucleic acid. (The wobble capture probesbind to the derivatized poly(dT)₁₄ when the Target Capture Reagent isprepared.)

To purify bound target nucleic acid, a DTS® 400 Target Capture System(Gen-Probe; Cat. No. 5210) was used to isolate and wash the magneticparticles. The DTS 400 Target Capture System has a test tube bay forpositioning the reaction tubes and applying a magnetic field thereto.The reaction tubes were placed in the test tube bay for about 3 minutesin the presence of the magnetic field to isolate the magnetic particleswithin the reaction tubes, after which the supernatants were aspirated.Each reaction tube was then provided with 1 mL of a Wash Buffer (10 mMHEPES, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v/v) ethanol, 0.02% (w/v)methylparaben, 0.01% (w/v) propylparaben, 150 mM NaCl, and 0.1% (w/v)sodium lauryl sulfate, adjusted to pH 7.5), covered with a sealing cardand vortexed for 15 seconds to resuspend the magnetic particles. Thereaction tubes were returned to the test tube bay and allowed to standat room temperature for 3 minutes before the Wash Buffer was aspirated.The wash steps were repeated once.

Following purification of the target nucleic acid, 75 FL of anAmplification/Detection Reagent (44.1 mM HEPES, 2.82% (w/v) trehalose,33 mM KCl, 0.01% (v/v) TRITON® X-100 detergent, 30.6 mM MgCl₂, 0.3%(v/v) ethanol, 0.1% methylparaben, 0.02% (w/v) propylparaben, 0.47 mMeach of dATP, dCTP, dGTP and dTTP, 1.76 mM each of rCTP and UTP, 9.41 mMrATP and 11.76 mM rGTP, adjusted to pH 7.7 at 23° C.) containing 11.9pmol/reaction of the T7 promoter-primer, 9.35 pmol/reaction of thenon-T7 primer, and 10 pmol/reaction of the molecular beacon probe wasadded to each reaction tube. The reaction tubes were covered with asealing card and mixed by vortexing for 15 seconds. After mixing, thecontents of the reaction tubes were transferred to separate reactionwells of a white, 96-well microplate (Thermo Electron Corporation,Waltham, Mass.; Product No. 9502887), each reaction well containing 75μL of an Oil Reagent (silicone oil (United Chemical Technologies, Inc.,Bristol, Pa.; Cat. No. PS038)). The microplate was covered with aThermalSeal film (Sigma-Aldrich Co., St. Louis, Mo.; Cat. No. Z369675)and incubated in a Solo HT Microplate Incubator (Thermo Electron; Cat.No. 5161580) at 60° C. for 5 minutes, and then in a Solo MicroplateIncubator (Thermo Electron; Cat. No. WI036) at 42° C. for 5 minutes.While in the second incubator, the sealing card was removed from themicrotiter plate and 25 μL of an Enzyme Reagent (58 mM HEPES, 50 mMN-acetyl-L-cysteine, 1.0 mM EDTA, 10% (v/v) TRITON® X-100 detergent, 3%(w/v) trehalose, 120 mM KCl, 20% (w/v) glycerol, 120 RTU/μL Moloneymurine leukemia virus reverse transcriptase (“MMLV-RT”), and 80 U/μL T7RNA polymerase, adjusted to pH 7.0) was added to each reaction well.(One reverse transcriptase unit (“RTU”) of activity for MMLV-RT isdefined as the incorporation of 1 nmol dTMP into DE81 filter-boundproduct in 20 minutes at 37° C. using (poly(rA)-p(dT)₁₂₋₁₈) as thesubstrate; and for T7 RNA polymerase, one unit (“U”) of activity isdefined as the production of 5.0 fmol RNA transcript in 20 minutes at37° C.) Immediately following addition of the Enzyme Reagent, thecontents of the reaction wells were mixed by stirring with standard 200μL pipette tips engaged by an 8-channel multi-pipettor and used totransfer the Enzyme Reagent to the microtiter plates. The microtiterplate was then re-sealed with a clear sealing card.

To detect the presence of amplification product in the reaction wells,the sealed plate was placed in a Fluoroskan Ascent® 100 MicroplateFluorometer (Thermo Electron; Product No. 5210480) pre-warmed to 42° C.and fluorescent readings were taken at 30-second intervals over a 50minute period. Detection depended upon a conformational change in themolecular beacon probes as they hybridized to amplification products,thereby resulting in the emission of detectable fluorescent signals. Aslong as the molecular beacon probes maintained a hairpin configuration,i.e., they were not hybridized to an amplification product of the targetnucleic acid, fluorescent emissions from the fluorescein reportermoieties were generally quenched by the DABCYL quencher moieties. But asmore of the molecular beacon probes hybridized to amplicon in thereaction wells, there was increase in detectable fluorescent signals.Thus, fluorescent emissions that increased over time provided anindication of active amplification of the target region of the targetnucleic acid. The results of this experiment are shown in FIG. 27, whereraw data from the fluorometer are plotted as fluorescent units (y-axis)versus time in minutes (x-axis) for each reaction well. Samplescontaining the target nucleic acid yielded strong fluorescent signalsthat emerged from background approximately 15 minutes into the reaction,while control samples yielded no significant signal above background.

Example 2 Automated Amplification Reactions in a Multi-Chambered,Flexible Receptacle Using Liquid Reagents

In this experiment, the TMA reaction of Section 1 of this example wasperformed using the receptacle 10 and instrument 100 illustrated inFIGS. 1A and 3. The receptacle 10 illustrated in FIG. 1B was pre-loadedwith reagents in the following manner: (i) 125 μL of the Target CaptureReagent was added to chamber C18; (ii) 3 mL of the Wash Buffer was addedto chamber C34; (iii) 25 μL of the Oil Reagent, followed by 85 μL of theAmplification/Detection Reagent, was added to chamber C20; and (iv) 35μL of the Oil Reagent, followed by 25 μL of the Enzyme Reagent, wasadded to chamber C32. After reagent loading, all of the chambers of thereceptacle 10 except chamber C16 were closed by heat sealing. A 500 μLtest sample having 10⁵ copies of the target nucleic acid, as describedin Example 1 above, was then pipetted into chamber C16, the samplechamber, which was then closed by heat sealing.

For the initial set-up, the sealed receptacle 10 was mounted on thefront portion 120 of the housing 104 and the door assembly 200 wasclosed, sandwiching the chambers of the receptacle 10 between thepressure mechanism cluster 180 and the thermal zones 260, 262, 264, 266,and 268 in the door assembly 200. The thermal zones were set to heatadjacent chambers at 30° C. The movement of materials between chambersof the receptacle 10 was controlled by the compression pads making upthe pressure mechanism cluster 180 described infra. Prior to startingthe test, compression pads P72, P70, P62, P51-1, P56, P58, P60, P64, P66and P68 were all activated to clamp and protect corresponding sealsassociated with portals (or neck) 72, 70, 62, 51, 56, 58, 60, 64, 66 and68, respectively, from prematurely opening or leaking Wash Buffer andair bubbles were removed from the vertical and lateral sections 42, 44of chamber C34, the wash buffer chamber, and the vertical inlet 48 ofchamber C36, the waster chamber, was concurrently closed by engaging thefollowing compression pads in the indicated order: (i) compression padsP34-1 and P36-1; (ii) compression pads P34-2 and P36-2; (iii)compression pads P34-3 and P36-3; (iv) compression pad P34-4; and (v)compression pad P34-5.

After the initial set-up, compression pad P68 was refracted andcompression pads P32 and P68 were sequentially activated to presschamber C32 and portal 68, thereby forcing open sealed portal 68 andmoving the Enzyme and Oil Reagents from chamber C32 to chamber C30. Atthe same time, compression pad P56 was retracted and compression padsP20 and P56 were sequentially activated to press chamber C20 and portal56, thereby forcing open sealed portal 56 and moving theAmplification/Detection and Oil Reagents from chamber C20 to chamberC22. Compression pads P68 and P56 remained activated to clamp portals 68and 56, respectively, thereby preventing a backflow of the Enzyme andAmplification/Detection Reagents into chambers C32 and C20.

After moving the Enzyme and Amplification/Detection Reagents,compression pads P18-1, P18-2 and P54 were sequentially activated topress chamber C18 and portal 54, thereby forcing open sealed portal 54and moving the Target Capture Reagent (“TCR”) from chamber C18 tochamber C16. The TCR and sample were mixed by twice moving the combinedcontents back-and-forth between chambers C16 and C18 using compressionpads associated with these chambers. Once mixing was completed,compression pad P54 was activated to clamp portal 54, therebymaintaining the TCR/sample mixture in chamber C16, where it wasincubated by heating thermal zone 260 at 30° C. for 5 minutes. Thisincubation step was carried out to facilitate non-specific binding ofthe target nucleic acid to the wobble capture probes and immobilizationof the wobble capture probes on the magnetically-responsive particlespresent in the TCR.

To separate the target nucleic acid from other material in the testsample, the magnet translation mechanism 208 was activated to move themagnet into position adjacent chamber C26 (referred to herein as the“on” position), the magnetic separation chamber 102 during the initialset-up. Compression pad P62 was retracted and compression pads P16-3,P16-4 and P62 were sequentially activated to press a portion of chamberC16, thereby forcing open sealed portal 62 and moving a first aliquot ofthe TCR/sample mixture from chamber C16 to chamber C26. After moving thefirst aliquot of the TCR/sample mixture to chamber C26, compression padP62 remained activated to clamp portal 62, thereby preventing themovement of material between chambers C16 and C26, and compression padsP16-3 and P16-4 were sequentially retracted. In chamber C26, themagnetically-responsive particles were subjected to the magnetic fieldsof the magnet for 1 minute at a temperature of 30° C. provided bythermal zone 268. While the magnet remained in the “on” position,compression pads P26, P70, P36-1, P36-2 and P36-3 were sequentiallyactivated to press chamber C26, portal 70 and the vertical inlet 48 ofchamber C36, the waste chamber, and to move liquid from chamber C26 intochamber C36.

By activating different arrangements of the compression pads associatedwith chambers C16 and C18, three additional aliquots of the TCR/samplemixture were moved from chambers C16 and C18 to chamber C26. For thesecond aliquot of TCR/sample mixture moved to chamber C26, thesequential operation of the compression pads was as follows: P18-1 (+),P18-2 (+), P18-2 (−), P62 (−), P16-3 (+), P16-4 (+), P62 (+), P16-3 (−)and P16-4 (−). For the third aliquot of TCR/sample mixture moved tochamber C26, the sequential operation of the compression pads was asfollows: P51-2 (+), P18-1 (−), P16-4 (+), P16-3 (+), P16-2 (+), P16-1(+), P16-1 (−), P16-2 (−), P16-3 (−), P16-4 (−), P18-1 (+), P18-2 (+),P54 (+), P62 (−), P16-3 (+), P16-4 (+), P62 (+), P16-3 (−) and P16-4(−). And for the fourth aliquot of TCR/sample mixture moved to chamberC26, the sequential operation of the compression pads was as follows:P16-2 (+), P16-1 (+), P62 (−), P16-3 (+), P16-4 (+), P16-3 (−) and P16-4(−). The (+) designation indicates that the referred to compression padwas activated to press a corresponding portal or portion of a chamber,and the (−) designation indicates that the referred to compression padwas retracted from corresponding portal or portion of a chamber. Theimmobilization and liquid waste removal steps were repeated for eachadditional aliquot until all of the TCR/sample mixture had beenprocessed and the sample reduced to a manageable size for furtherprocessing in the receptacle 10.

A wash procedure was then initiated to remove unwanted and potentiallyinterfering material from the immobilized nucleic acids, during whichthe magnet remained in the on position. At the start of the washprocedure, compression pads P34-5, P34-4, P34-3, P34-2 and P34-1 wereoperated to prime the vertical and lateral sections 42, 44 (the “neckregion”) of chamber C34 and, after retracting compression pad P72, topress on the neck region of chamber 34, thereby opening sealed portal 72and moving about 200 μL of Wash Buffer from chamber C34 to chamber C26.Compression pad P72 then clamped portal 72 and the Wash Buffer was movedback-and-forth three times between chamber C26 and the area covered bycompression pads P62 and P16-4 of chamber C16 by the action ofcompression pads P62, P16-4 and P26 to remove any residual TCR/samplemixture material lodged in opened portal 62 and to purify bound nucleicacids. In this step, P26 was only partially activated to preventoverfilling the areas of chamber C16 covered by compression pads P62 andP16-4 and to minimize foaming. All of the liquid was finally collectedin chamber C26 and exposed to the magnetic fields of the magnet for 1minute at a temperature of about 30° C. to immobilize anymagnetically-responsive particles. The Wash Buffer was then moved fromchamber C26 into chamber C36 by the action of compression pads P26, P70,P36-1, P36-2 and P36-3. Another aliquot of about 200 μL of Wash Bufferwas then moved from chamber C34 to chamber C26 by the operation ofcompression pads associated with the neck region of chamber C34,compression pad P72 was activated to clamp opened portal 72, and thewashing process was repeated, except that the Wash Buffer was only movedinto that portion of chamber C16 covered by compression pad P62, and themovement between chamber C26 and chamber C16 was only performed twice.Finally, a third aliquot of about 200 μL of Wash Buffer was moved fromchamber C34 to chamber C26 by the operation of compression padsassociated with the neck region of chamber C34, compression pad P72 wasactivated to clamp opened portal 72, and the magnetically-responsiveparticles were exposed to the magnetic fields of the magnet for 1 minuteat a temperature of 30° C. to immobilize any dislodgedmagnetically-responsive particles. Afterwards, the liquid was moved fromchamber C26 to chamber C34 by the action of compression pads P70, P36-1,P36-2 and P36-3. After the wash procedure was completed, the magnet wasmoved out of alignment with chamber C26 (the “off” position) and thermalzone 268 was moved into alignment with chamber C26.

Following separation of the target nucleic acid, compression pads P22and P58 were sequentially activated to press on chamber C22 and portal58, thereby forcing open sealed portal 58 and moving theAmplification/Detection Reagent from chamber C22 to chamber C26. Toensure that the magnetic particles were fully suspended in theAmplification/Detection Reagent, the Amplification/Detection Reagent wasmoved between chambers C22 and C26 two times by operation of compressionpads P26, P58 and P22. In chamber C26, the Amplification/DetectionReagent was incubated with thermal zone 268 at 62° C. (with a toleranceof, e.g., ±1.5° C.) for 5 minutes to facilitate binding of thepromoter-primer to target nucleic acids. At the same time, thermal zones264 and 266 brought the temperature of chambers C28 and C30 to 42° C.(with a tolerance of, e.g., ±0.45° C.), an optimal temperature for TMA.Following the 62° C. incubation, the contents of chamber C26 wereincubated at 42° C. for another 5 minutes, after which compression padsP26 and P64 were sequentially activated to press on chamber C26 andportal 64, thereby forcing open sealed portal 64 and moving the heatedAmplification/Detection Reagent and magnetic particle mixture fromchamber C26 to chamber C28. Once in chamber C28, compression pads P30and P66 were sequentially activated to press on chamber C30 and portal66, thereby forcing open sealed portal 66 and moving the heated EnzymeReagent from chamber C30 to chamber C28. After the Enzyme Reagent wasmoved to chamber C28, the temperature of thermal zone 266 was adjustedto 38° C.±1° C. To mix the Amplification/Detection Reagent, EnzymeReagent and magnetic particles, gravity assisted in draining thecontents of chamber C28 into chamber C30 through opened portal 66 andthen moved back into chamber C28 by sequentially pressing on chamber C30and portal 66 with compression pads P30 and P66. This process wasrepeated three times to ensure adequate mixing of the reagents foramplification of the target sequence, after which mixing compression padP66 was activated to clamp opened portal 66 and to move any residualreagents from opened portal 66 to chamber C28.

To detect amplification products generated in this mixture, thefluorometer 500 positioned adjacent a transparent window of chamber C28took fluorescent readings at 5-second intervals, each reading averagingjust over 4 seconds, during a 27 minute period. The results of thisexperiment are represented in FIG. 28, which is a graph showingfluorescence units detected from chamber C28 on the y-axis versus thetime in minutes on the x-axis. These results demonstrate that thereal-time TMA reaction performed using the instrument 100 and receptacle10 described herein gave equivalent results to the manually formattedreal-time TMA reaction of Example 1 above.

Example 3 Automated Amplification Reactions in a Multi-Chambered,Flexible Receptacle Using Liquid Reagents and a Urine Samples

This experiment was designed to evaluate a real-time TMA reaction usinga urine sample spiked with 10⁵ copies of target nucleic acid. Thematerials and methods of this experiment were the same as those of thereal-time TMA reaction described in Example 2, with the followingexceptions: (i) chamber C18 was loaded with 150 μL of the Target CaptureReagent containing 10 pmol of the wobble capture probe in combinationwith 100 μL of water; (ii) chamber C34 was loaded with 2 mL of the WashBuffer; (iii) the Oil Reagent was not loaded into chamber C20 or chamberC32 prior to loading the Amplification/Detection and Enzyme Reagentsinto these chambers; (iv) the test sample included 250 μL of urine froma healthy donor and 250 μL of the Sample Transport Medium; and (v) thesteps of moving the TCR/sample mixture from chamber C16 to chamber C26,subjecting the magnetic particles contained within chamber C26 to themagnetic fields of the magnet, and moving liquid from chamber C26 tochamber C36 while the magnetic particles were immobilized was repeatedonly two times. The results of this experiment are illustrated in FIG.29, which is a graph showing fluorescence units detected from chamberC28 on the y-axis versus time in minutes on the x-axis. These results ofthis experiment demonstrate that the real-time TMA reaction detected thetarget nucleic acid provided in the urine sample using the instrument100 and receptacle 10 described herein.

Example 4 Automated Amplifications Reaction in a Multi-Chambered,Flexible Receptacle Using Dried Reagents

The purpose of this experiment was to evaluate the automated, real-timeTMA reaction of Example 2 using dried forms of the Amplification andEnzyme/Probe Reagents. The receptacle 10 was pre-loaded with thefollowing reagents: (i) 125 μL of the Target Capture Reagent was addedto chamber C18; (ii) 3 mL of the Wash Buffer was added to chamber C34;(iii) 25 μL of the Oil Reagent, followed by 85 μL of an AmplificationReconstitution Reagent (0.4% (v/v) ethyl alcohol (absolute), 0.10% (w/v)methyl paraben, 0.02% (w/v) propyl paraben, 33 mM KCl, 30.6 mM MgCl₂,and 0.003% phenol red), was added to C-20; (iv) an Amplification ReagentPellet (formed from a 14 μL droplet containing 250 mM HEPES, 16% (w/v)trehalose, 53.4 mM ATP, 10 mM CTP, 66.6 mM GTP, 10 mM UTP, 2.66 mM ofeach of dATP, dCTP, dGTP and dTTP, adjusted to pH 7.0, 0.6 nmol/L of theantisense T7 promoter-primer and 0.47 nmol/L of the sense non-T7 primer,where the droplet was dispensed into liquid nitrogen and the resultingfrozen pellet was lyophilized) was added to chamber C22; (v) 35 μL ofthe Oil Reagent, followed by 25 μL of an Enzyme/Probe ReconstitutionReagent (50 mM HEPES, 1 mM EDTA, 10% (v/v) TRITON® X-100 detergent, and120 mM KCl, adjusted to pH 7.0), was added to chamber C32; and (vi) anEnzyme/Probe Reagent Pellet (formed from a 7.28 μL droplet containing 20mM HEPES, 125 mM N-acetyl-L-cysteine, 0.1 mM EDTA, 0.01% (v/v) TRITON®X-100 detergent, 20% (w/v) Trehalose, 412 MR/L MMLV-RT (dialyzed), 687MU/L T7 RNA polymerase (dialyzed), where “M” represents one million, and2.20 nmol/L of the molecular beacon probe) was added to chamber C30.After reagent loading, all of the chambers of the receptacle 10 exceptchamber C16 were closed by heat sealing. A 500 μL test sample having 10⁵copies of the target nucleic acid, as described in Example 1 above, wasthen pipetted into chamber C16, which was then closed by heat sealing.The initial set-up was the same as Example 2 above.

Following the initial set-up, compression pads P32 and P68 weresequentially activated to press on chamber C32 and portal 68, therebyforcing open sealed portal 68 and moving the Enzyme/Probe ReconstitutionReagent and Oil Reagent combination from chamber C32 to chamber C30,where the Enzyme/Probe Reagent Pellet was allowed to dissolve in theEnzyme/Probe Reconstitution Reagent for two minutes. Compression padsP20 and P56 were then sequentially activated to press on chamber C20 andportal P20, thereby forcing open sealed portal 56 and moving theAmplification Reconstitution Reagent and Oil Reagent combination fromchamber C20 to chamber C22, and compression pads P20 and P22 wereactivated to move the contents of chamber C22 back-and-forth four timesbetween chambers C20 and C22 to fully reconstitute the AmplificationReagent, after which compression pad P56 was activated to clamp portal56. Following a two minute dwell period in chamber C30, the compressionpads P30 and P32 were activated to move the contents of chamber C30back-and-forth two times between the chambers C30 and C32 to fullyreconstitute the Enzyme/Probe Reagent, after which compression pad P68was activated to clamp portal 68. The remainder of the steps were thesame as those Example 2.

The results of this experiment are illustrated in FIG. 30, which is agraph showing fluorescence units detected from chamber C28 on the y-axisversus the number of time in minutes on the x-axis. These results showthat this real-time TMA reaction, using pelleted amplification andenzyme/probe reagents that are reconstituted on-board, detected thetargeted transcript using the instrument 100 and receptacle 10 describedherein.

Example 5 Automated Amplification Reactions Using Liquid Reagents in thePresence or Absence of an Oil Reagent

This experiment was designed to evaluate the benefits of providing animmiscible liquid to open chambers of a multi-chambered receptacle priorto loading liquid reagents for performing TMA reactions. For thisexperiment, receptacles of the type illustrated in FIG. 4 were preparedin replicates of two as follows: (i) in the controls, no Oil Reagent wasadded to chambers containing the Amplification/Detection Reagent(chamber C20) or the Enzyme Reagent (chamber C32); (ii) 25 μL OilReagent was added to chamber C32 prior to adding the Enzyme Reagent andno oil was added to chamber C20; and (iii) 25 μL Oil Reagent was addedto each of chambers C20 and C32 prior to adding theAmplification/Detection and Enzyme Reagents. Chamber C18 was loaded with250 μL of the Target Capture Reagent containing 10 pmol of the wobblecapture probe and 100 μL of water. The materials and methods of thisexperiment were otherwise substantially the same as those of thereactions described in Example 2, except that the amplification reactionwas conducted at about 40° C. for 40 minutes. FIG. 31 is a graphillustrating the results of this experiment, showing the fluorescenceunits detected from chamber C28 on the y-axis versus time in minutes onthe x-axis. The results show that the real-time TMA reactions of thisexperiment performed noticeably better when at least one of theAmplification/Detection and Enzyme Reagents was combined with the OilReagent.

While the present invention has been described and shown in considerabledetail with reference to certain illustrative embodiments, those skilledin the art will readily appreciate other embodiments of the presentinvention. Accordingly, the present invention is deemed to include allmodifications and variations encompassed within the spirit and scope ofthe following appended claims.

1. A system for detecting electromagnetic radiation from the contents ofa receptacle, said system comprising: a receptacle holding areaconfigured to hold a receptacle; a receptacle held in said receptacleholding area; a detection element configured to receive electromagneticradiation and further adapted to generate a signal corresponding to acharacteristic of the electromagnetic radiation received by thedetection element; a transmission element disposed between saidreceptacle held in said receptacle holding area and said detectionelement and adapted to transmit electromagnetic radiation from thecontents of the receptacle to the detection element; a thermal elementin thermal conductivity with at least a portion of said transmissionelement and constructed and arranged to apply thermal energy to at leasta portion of said transmission element; and an actuator mechanismconstructed and arranged to move said transmission element between firstand second positions, wherein said transmission element is in greatercontact with said receptacle in the second position than in the firstposition, wherein said receptacle comprises a compressible portion thatis at least partially compressed by said transmission element when saidactuator moves said transmission element from the first position to thesecond position.
 2. The system of claim 1, wherein said detectionelement comprises a photodiode.
 3. The system of claim 1, furthercomprising an emission element adapted to emit electromagneticradiation.
 4. The system of claim 3, wherein said emission elementcomprises a light-emitting diode.
 5. The system of claim 1, wherein atleast a portion of said transmission element is configured to be incontact with at least a portion of the receptacle.
 6. The system ofclaim 1, wherein said transmission element comprises an optic elementadapted to transmit a light emission from the contents of thereceptacle.
 7. The system of claim 6, wherein said optic element is atransparent or translucent material.
 8. The system of claim 7, whereinsaid optic element is a plastic.
 9. The system of claim 6, wherein saidoptic element is adapted to transmit fluorescent light through at leasta portion of said optic element.
 10. The system of claim 1, wherein saidthermal element comprises an electrically-resistive film secured to asurface of said transmission element or embedded in at least a portionof said transmission element, said electrically-resistive film having anopening therein through which the electromagnetic radiation can betransmitted.
 11. The system of claim 1, wherein said transmissionelement comprises a peripheral wall defining a cavity, and wherein saidthermal element comprises an electrically-resistive film secured to asurface of said peripheral wall or embedded in at least a portion ofsaid peripheral wall for applying thermal energy to the space withinsaid cavity.
 12. The system of claim 1, wherein said thermal elementcomprises an electrically-resistive film secured to a surface of saidtransmission element or embedded in at least a portion of saidtransmission element, said electrically-resistive film being adapted totransmit the electromagnetic radiation through the resistive film. 13.The system of claim 1, wherein the electromagnetic radiation isfluorescence and said detection element is adapted to generate a signalcorresponding to the intensity of the fluorescence.
 14. The system ofclaim 1, wherein said transmission element is not in contact with saidreceptacle in the first position.
 15. The system of claim 1, whereinsaid compressible portion comprises a chamber formed from a flexiblematerial configured to yield to pressure applied externally to at leasta portion of said chamber.
 16. The system of claim 1, further comprisinga thermal element disposed adjacent to the receptacle holding area inopposed relationship to said transmission element.
 17. A method fordetecting electromagnetic radiation from the contents of a receptacle,said method comprising: transmitting electromagnetic radiation from thecontents of the receptacle to a detection element with a transmissionelement disposed between the receptacle and the detection element;applying thermal energy to the transmission element to cause atemperature of at least a portion of the transmission element to bedifferent from ambient temperature; detecting the electromagneticradiation with the detection element; moving the transmission elementbetween first and second positions, wherein said transmission element isin greater contact with the receptacle in the second position than inthe first position; and compressing at least a portion of the receptaclewith the transmission element when the transmission element is in thesecond position.
 18. The method of claim 17, wherein the step ofapplying thermal energy comprises heating a portion of the transmissionelement to a temperature above ambient temperature.
 19. The method ofclaim 17, further comprising causing the transmission element to be incontact with at least a portion of the receptacle.
 20. The method ofclaim 17, wherein the transmission element is not in contact with thereceptacle in the first position.
 21. The method of claim 17, whereinthe portion of the receptacle compressed with the transmission elementcomprises a chamber formed from a flexible material configured to yieldto pressure applied externally to at least a portion of said chamber, sothat compressing the chamber reduces an internal volume of the chamber.22. The method of claim 17, wherein the transmitting step comprisestransmitting a light emission from the contents of the receptacle. 23.The method of claim 22, wherein the detecting step comprises detecting afluorescent emission front the contents of the receptacle.
 24. Themethod of claim 23, further comprising directing excitation energy of aprescribed wavelength at the contents of the receptacle.
 25. The methodof claim 17, wherein the thermal energy is applied by a thermal elementin thermal contact with the transmission element.
 26. The method ofclaim 25, wherein the thermal element is in thermal contact with aportion of the transmission element through which the electromagneticradiation is transmitted to the detection element.
 27. The method ofclaim 25, wherein the thermal element is in thermal contact with aportion of a transmission element surrounding a portion of thetransmission element through which the electromagnetic radiation istransmitted to the detection element.
 28. The method of claim 25,wherein the transmission element comprises a peripheral wall defining acavity, and wherein the thermal element is in thermal contact with atleast a portion of the peripheral wall.
 29. The method of claim 25,wherein the thermal element is secured to a surface of the transmissionelement or embedded in at least a portion of the transmission element.30. The method of claim 17, further comprising applying thermal energyto the receptacle with a thermal element disposed adjacent thereceptacle and in opposed relationship to the transmission element. 31.The method of claim 17, wherein detecting the electromagnetic radiationcomprises detecting electromagnetic radiation of a prescribedwavelength.
 32. The method of claim 17, wherein detecting theelectromagnetic radiation comprises detecting the intensity of theelectromagnetic radiation.
 33. The method of claim 17, wherein thecontents of the receptacle are maintained at a substantially uniformtemperature.
 34. The method of claim 33, wherein the contents of thereceptacle are maintained at an essentially constant temperature duringsaid method.
 35. The method of claim 17, further comprising performingan amplification reaction in the receptacle during said method.
 36. Adevice adapted to transmit electromagnetic radiation and to applythermal energy to a receptacle, said device comprising: a transmissionelement adapted to transmit a light emission through at least a portionof said transmission element; a thermal element secured to a surface ofsaid transmission element or embedded in at least a portion of saidtransmission element and constructed and arranged to apply thermalenergy to at least a portion of said transmission element to therebyraise or lower an outer surface temperature of said transmissionelement; and an actuator mechanism constructed and arranged to move saidtransmission element between first and second positions with respect toa receptacle disposed adjacent said transmission element, such that saidtransmission element will be in greater contact with the receptacle inthe second position than in the first position, and whereby thereceptacle will be at least partially compressed by said transmissionelement when said actuator moves said transmission element from thefirst position to the second position.
 37. The device of claim 36,wherein said transmission element is a transparent or translucentmaterial.
 38. The device of claim 37, wherein said transmission elementis a plastic.
 39. The device of claim 36, wherein said transmissionelement is adapted to transmit fluorescent light through at least aportion of said optic element.
 40. The device of claim 36, wherein saidthermal element comprises an electrically-resistive film secured to asurface of said transmission element or embedded in at least a portionof said transmission element, said electrically-resistive film having anopening therein through which electromagnetic radiation can betransmitted.
 41. The device of claim 36, wherein said transmissionelement comprises a peripheral wall defining a cavity, and wherein saidthermal element comprises an electrically-resistive film secured to asurface of said peripheral wall or embedded in at least a portion ofsaid peripheral wall for applying thermal energy to the space withinsaid cavity.
 42. The device of claim 36, wherein said thermal elementcomprises an electrically-resistive film secured to a surface of saidtransmission element or embedded in at least a portion of saidtransmission element, said electrically-resistive film being adapted totransmit electromagnetic radiation through the resistive film.